JP2020130055A - Endometrium-like tissue and method for producing the same - Google Patents
Endometrium-like tissue and method for producing the same Download PDFInfo
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- JP2020130055A JP2020130055A JP2019028336A JP2019028336A JP2020130055A JP 2020130055 A JP2020130055 A JP 2020130055A JP 2019028336 A JP2019028336 A JP 2019028336A JP 2019028336 A JP2019028336 A JP 2019028336A JP 2020130055 A JP2020130055 A JP 2020130055A
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Abstract
Description
本発明は、子宮内膜様組織及びその製造方法に関する。 The present invention relates to endometrial-like tissue and a method for producing the same.
近年、女性の社会進出による晩婚化の影響で、さまざまな産科疾患を併発しやすい高齢妊娠が増加している。また、不妊症と診断される患者も増加しており、社会問題となっている。 In recent years, due to the effects of late marriage due to the advancement of women into society, the number of elderly pregnancies that are prone to complications of various obstetric diseases is increasing. In addition, the number of patients diagnosed with infertility is increasing, which has become a social problem.
不妊症の原因は女性因子と男性因子に分けられるが、女性因子においてはさらに、***因子、卵管因子、子宮因子、頸管因子、免疫学的因子などに分けられる。これらの不妊症に対する治療法として、様々な生殖補助医療（ＡＲＴ）や、それ以外の治療法が開発されているが、未だ十分な治療法とはいえない。 The causes of infertility are divided into female factors and male factors, and female factors are further divided into ovulation factors, fallopian tube factors, uterine factors, cervical factors, immunological factors and the like. Various assisted reproductive technology (ART) and other treatments have been developed as treatments for these infertility, but they are not yet sufficient treatments.
子宮因子による不妊症は、子宮内腔および子宮内膜の変化が原因であるといわれている。子宮内腔の癒着・変性を伴う疾患の治療法として、子宮組織の再生を目指した再生医療技術が開発されつつある。例えば、子宮内膜上皮細胞シート・子宮内膜間質細胞シートを作製し、それらを積層化してラットの子宮へ移植すると、子宮内膜が再生し、妊娠が成立することが報告されている(特許文献１）。 Infertility due to uterine factors is said to be caused by changes in the uterine cavity and endometrium. Regenerative medicine technology aimed at the regeneration of uterine tissue is being developed as a treatment method for diseases associated with adhesion and degeneration of the uterine cavity. For example, it has been reported that when endometrial epithelial cell sheets and endometrial stromal cell sheets are prepared, laminated and transplanted into the uterus of rats, the endometrium is regenerated and pregnancy is established ( Patent Document 1).
生殖補助医療においては、***誘発法や胚培養法が年々改善されており、胚移植率が向上してきたが、良好な受精卵を子宮へ移植しても一定の確率で着床しないなどの問題が未だに残っている。そのため、受精卵の着床率を向上するために、子宮内膜と胚との因果関係を明らかにすることが求められている。 In assisted reproductive technology, the ovulation induction method and embryo culture method have been improved year by year, and the embryo transfer rate has improved, but there are problems such as implantation of a good fertilized egg into the uterus with a certain probability. Still remains. Therefore, in order to improve the implantation rate of fertilized eggs, it is required to clarify the causal relationship between the endometrium and the embryo.
子宮内膜と胚との因果関係を明らかにするために行われたこれまでのインビトロ研究は、単層の培養子宮内膜細胞などの薄い構造体を用いた研究であり（非特許文献１）、受精卵が浸潤する過程を観察可能なインビトロモデルは未だ開発されていない。 Previous in vitro studies conducted to clarify the causal relationship between the endometrium and the embryo are studies using thin structures such as single-layer cultured endometrial cells (Non-Patent Document 1). , An in vitro model capable of observing the infiltration process of fertilized eggs has not yet been developed.
本発明は、インビトロにおいて、受精卵移植に伴う子宮組織に起こる変化を観察可能とする子宮内膜様組織及びその製造方法を提供することを目的とする。 An object of the present invention is to provide an endometrial-like tissue and a method for producing the same, which enables observation of changes in the uterine tissue associated with fertilized egg transplantation in vitro.
本発明者らは、種々の角度から検討を加えて研究開発を行ってきた。その結果、
細胞接着性ハイドロゲルを含む支持体と、前記支持体の上に形成された、子宮内膜間質細胞を含む第一細胞層と、前記第一細胞層の上に形成された、子宮内膜上皮細胞を含む第二細胞層とを含む子宮内膜様組織は、驚くべきことに、インビトロにおいて受精卵移植後の変化を観察できることを見出し、本発明を開発するに至った。
The present inventors have conducted research and development by examining from various angles. as a result,
A support containing cell-adhesive hydrogel, a first cell layer containing endometrial stromal cells formed on the support, and an endometrium formed on the first cell layer. Surprisingly, it was found that the endometrial tissue containing the second cell layer containing epithelial cells can observe changes after fertilized egg transplantation in vitro, leading to the development of the present invention.
すなわち、本発明は、以下の態様を含んでいる。 That is, the present invention includes the following aspects.
［１］ 細胞接着性ハイドロゲルを含む支持体と、
前記支持体の上に形成された、子宮内膜間質細胞を含む第一細胞層と、
前記第一細胞層の上に形成された、子宮内膜上皮細胞を含む第二細胞層と
を含む、子宮内膜様組織。
［２］ 前記細胞接着性ハイドロゲルが、コラーゲンを含む、［１］に記載の子宮内膜様組織。
［３］ 前記細胞接着性ハイドロゲルが、フィブリンを含む、［１］または［２］に記載の子宮内膜様組織。
［４］ 前記第一細胞層が、２層以上形成されている、［１］〜［３］のいずれか１項に記載の子宮内膜様組織。
［５］ 前記子宮内膜間質細胞が、初代子宮内膜間質細胞である、［１］〜［４］のいずれか１項に記載の子宮内膜様組織。
［６］ 前記子宮内膜上皮細胞が、初代子宮内膜上皮細胞である、［１］〜［５］のいずれか１項に記載の子宮内膜様組織。
［７］ 前記第二細胞層の上に受精卵が適用されている、［１］〜［６］のいずれか１項に記載の子宮内膜様組織。
[1] A support containing a cell-adhesive hydrogel and
A first cell layer containing endometrial stromal cells formed on the support,
Endometrial-like tissue formed on the first cell layer and comprising a second cell layer containing endometrial epithelial cells.
[2] The endometrial-like tissue according to [1], wherein the cell-adhesive hydrogel contains collagen.
[3] The endometrial-like tissue according to [1] or [2], wherein the cell-adhesive hydrogel contains fibrin.
[4] The endometrial-like tissue according to any one of [1] to [3], wherein two or more layers of the first cell layer are formed.
[5] The endometrial-like tissue according to any one of [1] to [4], wherein the endometrial stromal cells are primary endometrial stromal cells.
[6] The endometrial-like tissue according to any one of [1] to [5], wherein the endometrial epithelial cells are primary endometrial epithelial cells.
[7] The endometrial-like tissue according to any one of [1] to [6], wherein a fertilized egg is applied on the second cell layer.
［８］ 子宮内膜様組織を製造する方法であって、以下：
（１）細胞接着性ハイドロゲルを含む支持体の上に、子宮内膜間質細胞を含む第一細胞層と、子宮内膜上皮細胞を含む第二細胞層とを形成する工程であって、ここで前記第二細胞層は、前記第一細胞層の上に形成される、工程；および
（２）前記工程（１）で得られる構築物を培養する工程、
を含む、方法。
［９］ 前記細胞接着性ハイドロゲルが、コラーゲンを含む、［８］に記載の方法。
［１０］ 前記細胞接着性ハイドロゲルが、フィブリンを含む、［８］または［９］に記載の方法。
［１１］ 前記第一細胞層を、２層以上形成されている、［８］〜［１０］のいずれか１項に記載の方法。
［１２］ 前記工程（１）が、
前記第一細胞層の上に前記第二細胞層を形成した積層体を、前記支持体の上にのせる工程である、［８］〜［１１］のいずれか１項に記載の方法。
［１３］ 前記第一細胞層および前記第二細胞層が、刺激応答性培養基材を用いることにより得られる、［８］〜［１２］のいずれか１項に記載の方法。
［１４］ 前記刺激応答性培養基材が、温度応答性培養基材である、［１３］に記載の方法。
［１５］ 前記子宮内膜間質細胞が、初代子宮内膜間質細胞である、［８］〜［１４］のいずれか１項に記載の方法。
［１６］ 前記子宮内膜上皮細胞が、初代子宮内膜上皮細胞である、［８］〜［１５］のいずれか１項に記載の方法。
［１７］ （３）前記第二細胞層の上に受精卵を適用する、［８］〜［１６］のいずれか１項に記載の方法。
[8] A method for producing endometrial-like tissue, which includes the following:
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Including methods.
[9] The method according to [8], wherein the cell adhesion hydrogel contains collagen.
[10] The method according to [8] or [9], wherein the cell adhesion hydrogel contains fibrin.
[11] The method according to any one of [8] to [10], wherein two or more layers of the first cell layer are formed.
[12] The step (1)
The method according to any one of [8] to [11], which is a step of placing the laminate having the second cell layer formed on the first cell layer on the support.
[13] The method according to any one of [8] to [12], wherein the first cell layer and the second cell layer are obtained by using a stimulus-responsive culture substrate.
[14] The method according to [13], wherein the stimulus-responsive culture substrate is a temperature-responsive culture substrate.
[15] The method according to any one of [8] to [14], wherein the endometrial stromal cells are primary endometrial stromal cells.
[16] The method according to any one of [8] to [15], wherein the endometrial epithelial cells are primary endometrial epithelial cells.
[17] (3) The method according to any one of [8] to [16], wherein the fertilized egg is applied on the second cell layer.
［１８］ ［８］〜［１７］のいずれか１項に記載の方法により得られる子宮内膜様組織。 [18] Endometrial-like tissue obtained by the method according to any one of [8] to [17].
［１９］ ［１］〜［７］および［１８］のいずれか１項に記載の子宮内膜様組織を用いた、受精卵の着床を促進する因子をスクリーニングする方法。 [19] A method for screening a factor that promotes implantation of a fertilized egg using the endometrial-like tissue according to any one of [1] to [7] and [18].
本発明の子宮内膜様組織により、受精卵移植後に子宮組織に起こる変化をインビトロにおいて観察可能となる。 The endometrial-like tissue of the present invention allows in vitro observation of changes that occur in the uterine tissue after fertilized egg transplantation.
以下、発明の実施の形態を通じて本発明を説明するが、以下の実施形態は特許請求の範囲にかかる発明を限定するものではなく、また実施形態の中で説明されている特徴の組み合わせの全てが発明の解決手段に必須であるとは限らない。なお、以下の「子宮内膜様組織」、「子宮内膜様組織を製造する方法」または「子宮内膜組織モデルを用いた受精卵の着床を促進する因子をスクリーニングする方法」で説明されている事項は、「子宮内膜様組織」、「子宮内膜様組織を製造する方法」および「子宮内膜組織モデルを用いた受精卵の着床を促進する因子をスクリーニングする方法」のいずれにおいても適用され得る。 Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention in the claims, and all combinations of features described in the embodiments are included. It is not always essential for the means of solving the invention. In addition, it is explained in the following "endometrial tissue", "method for producing endometrial tissue" or "method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model". The items are either "endometrial tissue", "method for producing endometrial tissue", or "method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model". Can also be applied in.
本明細書において、「第一」「第二」等の用語は、１つの要素をもう１つの要素と区別するために用いられるものであり、例えば、第一の要素を第二の要素と表現し、同様に第二の要素を第一の要素と表現してもよく、これによって本発明の範囲を逸脱するものではない。 In the present specification, terms such as "first" and "second" are used to distinguish one element from another, and for example, the first element is expressed as the second element. Similarly, the second element may be expressed as the first element, which does not deviate from the scope of the present invention.
＜＜子宮内膜様組織＞＞
本発明は、細胞接着性ハイドロゲルを含む支持体と、
前記支持体の上に形成された、子宮内膜間質細胞を含む第一細胞層と、
前記第一細胞層の上に形成された、子宮内膜上皮細胞を含む第二細胞層と
を含む、子宮内膜様組織を提供する。また、本発明は、後述の子宮内膜様組織を製造する方法によって得られる子宮内膜様組織を提供する。
<< Endometrial tissue >>
The present invention includes a support containing a cell-adhesive hydrogel and
A first cell layer containing endometrial stromal cells formed on the support,
Provided is an endometrial-like tissue formed on the first cell layer and containing a second cell layer containing endometrial epithelial cells. The present invention also provides endometrial-like tissue obtained by the method for producing endometrial-like tissue described later.
本明細書において、「子宮内膜様組織」とは、インビトロにおいて再構築され、生体の子宮内膜の構造を模倣した構造体をいうが、生体の子宮内膜が有さない構成要素を含むものであってもよい。 As used herein, the term "endometrium-like tissue" refers to a structure that is reconstructed in vitro and mimics the structure of the endometrium of a living body, but includes components that the endometrium of a living body does not have. It may be a thing.
本明細書において、「細胞接着性ハイドロゲル」とは、細胞外マトリックスを構成するタンパク質、或いは、キトサンゲル、コラーゲンゲル、ゼラチン、ペプチドゲル、ラミニンゲル及びフィブリンゲル、並びにそれらの混合物からなる群から選択されるものをいう。これらのうち、キトサンゲル、コラーゲンゲル、ゼラチンゲル、ペプチドゲル及びラミニンゲルは、例えば温度、ｐＨ及び／又は塩濃度を変更することによりゲル化が促進される。フィブリンゲルは、モノマーであるフィブリノゲンが、酵素であるトロンビンと作用することによりゲル化される。本発明に用いられる細胞接着性ハイドロゲルは、好ましくはコラーゲンゲルおよび／またはフィブリンゲルであり、より好ましくは、コラーゲンゲルとフィブリンゲルの混合物である。本明細書において、コラーゲンゲルに含まれるコラーゲンとは、例えば、タイプＩ、タイプＩＩ、タイプＩＩＩ、タイプＩＶ、タイプＶ、タイプＶＩ、タイプＶＩＩ、タイプＶＩＩＩ、タイプＩＸ、タイプＸ、タイプＸＩ、タイプＸＶ、タイプＸＶＩＩ、もしくはタイプＸＶＩＩＩのコラーゲン又はアテロコラーゲン、あるいはその組み合わせである。コラーゲンゲルに含まれるコラーゲンは、例えば、哺乳動物由来（例えば、霊長類（ヒト及びヒト以外の霊長類を含む）、げっ歯類（マウス、ラット、ハムスター、モルモット等）、ウサギ、イヌ、ウシ、ウマ、ブタ、ネコ、ヤギ、ヒツジ等）であってもよく、哺乳動物のコラーゲン遺伝子を基に、遺伝子工学的に得られたものであってもよい。 As used herein, the term "cell adhesion hydrogel" is selected from the group consisting of proteins constituting extracellular matrix, chitosan gel, collagen gel, gelatin, peptide gel, laminin gel and fibrin gel, and mixtures thereof. It means what is done. Of these, chitosan gel, collagen gel, gelatin gel, peptide gel and laminin gel are promoted to gel by changing the temperature, pH and / or salt concentration, for example. Fibrin gel is gelled by the action of the monomer fibrinogen with the enzyme thrombin. The cell adhesion hydrogel used in the present invention is preferably a collagen gel and / or a fibrin gel, and more preferably a mixture of a collagen gel and a fibrin gel. In the present specification, the collagen contained in the collagen gel is, for example, type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type. XV, type XVII, or type XVIII collagen or atelocollagen, or a combination thereof. The collagen contained in the collagen gel is, for example, derived from mammals (for example, primates (including humans and non-human primates)), rodents (mouse, rat, hamster, guinea pig, etc.), rabbits, dogs, cows, etc. It may be a horse, a pig, a cat, a goat, a sheep, etc.), or it may be obtained by genetic engineering based on a mammalian collagen gene.
本明細書において、「支持体」とは、後述の「細胞層（第一細胞層および第二細胞層）」を接着させて固定するために用いられる構造体をいう。本発明において用いられる支持体は、細胞接着性ハイドロゲルを含み、それにより細胞層との接着が促進される。本発明において用いられる支持体は、例えば、細胞接着性ハイドロゲルがコーティングされた培養基材（例えば、培養容器）またはセルカルチャーインサートであってもよく、細胞接着性ハイドロゲル自体であってもよい。細胞接着性ハイドロゲルを支持体として用いる場合、例えば、硬化する前の細胞接着性ハイドロゲル溶液をモールド（フレーム）に流し込み、所望の形状に硬化させた支持体を用いてもよい。例えば、国際公開第２０１２／０３６２２５に記載の方法により、支持体を形成してもよい。また、支持体には、国際公開第２０１２／０３６２２５に記載されるような、培地が流れる流路が設けられていてもよい。 As used herein, the term "support" refers to a structure used for adhering and fixing "cell layers (first cell layer and second cell layer)" described later. The support used in the present invention contains a cell-adhesive hydrogel, which promotes adhesion to the cell layer. The support used in the present invention may be, for example, a culture substrate (for example, a culture vessel) coated with a cell-adhesive hydrogel or a cell culture insert, or may be a cell-adhesive hydrogel itself. .. When the cell adhesion hydrogel is used as a support, for example, a support in which a cell adhesion hydrogel solution before curing is poured into a mold (frame) and cured into a desired shape may be used. For example, the support may be formed by the method described in International Publication No. 2012/036225. In addition, the support may be provided with a flow path through which the medium flows, as described in International Publication No. 2012/036225.
一実施態様において、細胞接着性ハイドロゲルを含む支持体として用いられ得る「コラーゲンゲルとフィブリンゲルの混合物」は、細胞層を接着させて培養した後においても細胞層が剥離しないように、その含有物の量を調整すればよい。硬化前のコラーゲン／フィブリン溶液に含まれるコラーゲン対フィブリノゲンの比は、例えば、約１０：約１〜約１：約１０であってもよく、約５：約１〜約１：約５であってもよく、約１：約３であってもよってもよい。本明細書において、「約」とは、その数値の±１０％の範囲に含まれる数値を意味する。 In one embodiment, a "mixture of collagen gel and fibrin gel" that can be used as a support containing a cell-adhesive hydrogel contains the cell layer so that the cell layer does not peel off even after the cell layer is adhered and cultured. You just have to adjust the amount of things. The ratio of collagen to fibrinogen contained in the collagen / fibrin solution before curing may be, for example, about 10: about 1 to about 1: about 10, and about 5: about 1 to about 1: about 5. It may be about 1: about 3. As used herein, the term "about" means a numerical value included in the range of ± 10% of the numerical value.
本明細書において、「子宮組織」とは、哺乳類のメスの生殖器であり、妊娠時に体内で胎児を育てる器官をいう。哺乳類、特にヒトにおける子宮組織は、箱状の腔を形成している。子宮体部において受精卵の発生、胎児の育成を行う。子宮体部は、子宮内膜、筋層、漿膜から構成されている。 As used herein, the term "uterine tissue" refers to the female reproductive organs of mammals and the organs that raise the fetus in the body during pregnancy. Uterine tissue in mammals, especially humans, forms a box-shaped cavity. Fertilized eggs are generated and the fetus is raised in the uterine body. The body of the uterus is composed of the endometrium, muscularis, and serosa.
子宮内膜は、組織構造の観点から、子宮内膜上皮層と子宮内膜間質層に分類できる。子宮内膜上皮層は、単層円柱の細胞からなり、分泌細胞と線毛細胞を含み、子宮腔にもっとも近い細胞層である。子宮内膜間質層は、子宮内膜腺を数多く有した構造である。子宮内膜線の入り口は子宮腔に面した上皮とつながっており、一番奥は子宮内膜の深部まで伸びている。子宮内膜腺からは、粘液が分泌され子宮内膜の表面を覆い、受精卵・胚の発育を助ける。 The endometrium can be classified into an endometrial epithelial layer and an endometrial stromal layer from the viewpoint of tissue structure. The endometrial epithelial layer consists of simple columnar cells, contains secretory cells and ciliated cells, and is the cell layer closest to the uterine cavity. The endometrial stromal layer is a structure with many endometrial glands. The entrance of the endometrium line is connected to the epithelium facing the uterine cavity, and the innermost part extends to the deep part of the endometrium. Mucus is secreted from the endometrium glands to cover the surface of the endometrium and support the development of fertilized eggs and embryos.
子宮内膜は、機能面の観点から、性周期で変化が起こらない子宮内膜の深部にある基底層と、性周期で厚さ及び構造を変化させ、子宮内膜腺やラセン動脈を有する機能層の２つに分類することができる。 From a functional point of view, the endometrium has a basal layer in the deep part of the endometrium that does not change in the sexual cycle, and a function that changes the thickness and structure in the sexual cycle and has endometrial glands and helicine arteries. It can be divided into two layers.
ヒトの子宮内膜は、女性ホルモンであるエストロゲン、プロゲステロンに依存し、性周期に応じてその厚さを変化させる。月経終了から***期までの期間、エストロゲンのレベルが増加し、子宮内膜、特に機能層が増殖する（増殖期）。***期を過ぎると一時的にエストロゲンのレベルが減少し、その後、再びエストロゲン及びプロゲステロンのレベルが増加し、子宮内膜が肥厚化する（分泌期）。このとき、子宮内膜の子宮腺が発達し、分泌が促進され、子宮内膜間質層の浮腫が起きる。こうして受精卵の着床に適した環境が作りだされる。その後、平均して２８日周期で月経期となり、機能層の剥離が起こる。この時、エストロゲン、プロゲステロンのレベルはともに減少する。性周期による子宮粘膜の肥厚化と剥離は、女性において正常な妊娠を行う上で重要な役割を果たしている。 The human endometrium depends on the female hormones estrogen and progesterone, and its thickness changes according to the sexual cycle. During the period from the end of menstruation to the ovulation period, estrogen levels increase and the endometrium, especially the functional layer, proliferates (proliferation phase). After the ovulation period, estrogen levels temporarily decrease, then estrogen and progesterone levels increase again, and the endometrium thickens (secretory period). At this time, the uterine gland of the endometrium develops, secretion is promoted, and edema of the endometrial stroma occurs. In this way, an environment suitable for implantation of fertilized eggs is created. After that, the menstrual period occurs on average every 28 days, and the functional layer peels off. At this time, both estrogen and progesterone levels decrease. Thickening and detachment of the uterine mucosa due to the sexual cycle plays an important role in normal pregnancy in women.
本明細書において、「子宮内膜間質細胞」とは、子宮内膜上皮細胞の支持組織を構成する細胞をいい、子宮内膜間質層に含まれる細胞をいう。本発明に用いられる子宮内膜間質細胞は、子宮内膜組織より単離された初代子宮内膜間質細胞であってもよく、株化された子宮内膜間質細胞でもよく、ＥＳ細胞、ｉＰＳ細胞、Ｍｕｓｅ細胞等の多能性幹細胞から誘導された子宮内膜間質細胞であってもよい。好ましくは、本発明に用いられる子宮内膜間質細胞は初代子宮内膜間質細胞である。 As used herein, the term "endometrial stromal cell" refers to a cell that constitutes a supporting tissue of endometrial epithelial cells, and refers to a cell contained in the endometrial stromal layer. The endometrial stromal cells used in the present invention may be primary endometrial stromal cells isolated from endometrial tissue, or established endometrial stromal cells, or ES cells. , IPS cells, Muse cells and other pluripotent stem cells may be endometrial stromal cells. Preferably, the endometrial stromal cells used in the present invention are primary endometrial stromal cells.
一実施態様において、本発明にかかる「子宮内膜間質細胞を含む第一細胞層」は、子宮内膜間質細胞以外の細胞が含まれていてもよく、その細胞の種類は特に限定されない。また、子宮内膜間質細胞は単一の種類の細胞であってもよく、複数の種類の細胞が含まれてもよい。一実施態様において、本発明にかかる第一細胞層に含まれる子宮内膜間質細胞の割合は、６０％以上、好ましくは７０％以上、さらに好ましくは８０％以上（例えば、８０％、８５％、９０％、９５％、９６％、９７％、９８％、９９％、１００％）であり得る。 In one embodiment, the "first cell layer containing endometrial stromal cells" according to the present invention may contain cells other than endometrial stromal cells, and the type of the cells is not particularly limited. .. In addition, the endometrial stromal cells may be a single type of cell or may contain a plurality of types of cells. In one embodiment, the proportion of endometrial stromal cells contained in the first cell layer according to the present invention is 60% or more, preferably 70% or more, still more preferably 80% or more (for example, 80%, 85%). , 90%, 95%, 96%, 97%, 98%, 99%, 100%).
本明細書において、「子宮内膜上皮細胞」は、子宮粘膜（子宮内膜）に含まれる上皮細胞であり、子宮内膜上皮層に含まれる細胞をいう。本発明に用いられる子宮内膜上皮細胞は、子宮内膜組織より単離された初代子宮内膜上皮細胞であってもよく、株化された子宮内膜上皮細胞でもよく、ＥＳ細胞、ｉＰＳ細胞、Ｍｕｓｅ細胞等の多能性幹細胞から誘導された子宮内膜上皮細胞であってもよい。好ましくは、本発明に用いられる子宮内膜上皮細胞は初代子宮内膜上皮細胞である。 In the present specification, the "endometrial epithelial cell" is an epithelial cell contained in the uterine mucosa (endometrium) and refers to a cell contained in the endometrial epithelial layer. The endometrial epithelial cells used in the present invention may be primary endometrial epithelial cells isolated from endometrial tissue, or established endometrial epithelial cells, ES cells, iPS cells. , Endometrial epithelial cells derived from pluripotent stem cells such as Muse cells. Preferably, the endometrial epithelial cells used in the present invention are primary endometrial epithelial cells.
一実施態様において、本発明にかかる「子宮内膜上皮細胞を含む第二細胞層」は、子宮内膜上皮細胞以外の細胞が含まれていてもよく、その細胞の種類は特に限定されない。また、子宮内膜上皮細胞は単一の種類の細胞であってもよく、複数の種類の細胞が含まれてもよい。一実施態様において、本発明にかかる第二細胞層に含まれる子宮内膜上皮細胞の割合は、６０％以上、好ましくは７０％以上、さらに好ましくは８０％以上（例えば、８０％、８５％、９０％、９５％、９６％、９７％、９８％、９９％、１００％）であり得る。 In one embodiment, the "second cell layer containing endometrial epithelial cells" according to the present invention may contain cells other than endometrial epithelial cells, and the type of the cells is not particularly limited. In addition, the endometrial epithelial cells may be a single type of cell or may contain a plurality of types of cells. In one embodiment, the proportion of endometrial epithelial cells contained in the second cell layer according to the present invention is 60% or more, preferably 70% or more, still more preferably 80% or more (for example, 80%, 85%, etc.). It can be 90%, 95%, 96%, 97%, 98%, 99%, 100%).
本発明に用いられる細胞の動物種の由来は、特に制約されないが、例えば、ヒト、或いはラット、マウス、モルモット、マーモセット、ウサギ、イヌ、ネコ、ヒツジ、ブタ、ヤギ、サル、チンパンジーあるいはそれらの免疫不全動物等が挙げられる。また、適用する受精卵を採取した同一の個体由来の細胞であってもよく、受精卵を採取した個体とは別の個体由来の細胞であってもよい。また、子宮内膜細胞の由来動物と受精卵の由来動物が必ずしも一致する必要もない。また、子宮内膜様組織を構成する細胞の由来動物と、受精卵の由来動物とは、必ずしも一致しなくてもよい。 The origin of the animal species of the cells used in the present invention is not particularly limited, but for example, humans or rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees or their immunity. Examples include incomplete animals. Further, it may be a cell derived from the same individual from which the fertilized egg to be applied is collected, or a cell derived from an individual different from the individual from which the fertilized egg was collected. In addition, the animal from which the endometrial cell is derived and the animal from which the fertilized egg is derived do not necessarily have to match. In addition, the animal from which the cells constituting the endometrial tissue are derived and the animal from which the fertilized egg is derived do not necessarily have to match.
本明細書において、「細胞層（第一細胞層および第二細胞層）」とは、シート状の細胞群をいい、「細胞シート」ともいう。一実施態様において、本発明の子宮内膜様組織に含まれる細胞層（第一細胞層または第二細胞層）は、１細胞の厚さを有する細胞層であってもよいが、収縮して１細胞の厚さを超えた厚さ（例えば、２〜１０細胞分の厚さ）を有する細胞層であることが好ましい。接着していた足場から剥離され、収縮した細胞層（例えば、収縮した細胞シート）は、隣接した細胞の間に形成された細胞結合（例えば、タイトジャンクションやギャップジャンクションなど）が維持され、かつ、細胞密度も高いため、単層の細胞層よりも種々の機能を発揮する。 In the present specification, the "cell layer (first cell layer and second cell layer)" refers to a sheet-like cell group, and is also referred to as a "cell sheet". In one embodiment, the cell layer (first cell layer or second cell layer) contained in the endometrial tissue of the present invention may be a cell layer having a thickness of one cell, but contracts. It is preferable that the cell layer has a thickness exceeding the thickness of one cell (for example, a thickness of 2 to 10 cells). The cell layer that has been detached from the adhered scaffold and contracted (for example, a contracted cell sheet) maintains the cell junctions (for example, tight junctions and gap junctions) formed between adjacent cells, and Due to its high cell density, it exerts more functions than a single-layer cell layer.
細胞層を得る方法としては、例えば、温度、ｐＨ、光、電気等の刺激によって分子構造を変化させる高分子が被覆された刺激応答性培養基材の上で細胞を培養し、温度、ｐＨ、光、電気等の刺激の条件を変えて刺激応答性培養基材の表面の性質を変化させることで、細胞同士の接着状態は維持しつつ、刺激応答性培養基材から細胞をシート状に剥離する方法；任意の培養基材の上で細胞を培養し、シート状の細胞群を端部から物理的にピンセット等により剥離する方法；そして、ハイドロゲル等と共に細胞を培養表面上に播種し、形成されたシート状の細胞群を剥離する方法、などが挙げられるが、これらに限定されない。 As a method for obtaining a cell layer, for example, cells are cultured on a stimulus-responsive culture medium coated with a polymer that changes the molecular structure by stimuli such as temperature, pH, light, and electricity, and the temperature, pH, By changing the surface properties of the stimulus-responsive culture substrate by changing the stimulus conditions such as light and electricity, the cells are separated from the stimulus-responsive culture substrate into a sheet while maintaining the adhered state between the cells. A method of culturing cells on an arbitrary culture medium and physically peeling the sheet-shaped cell group from the end with a tweezers or the like; and seeding the cells on the culture surface together with hydrogel or the like. Examples thereof include, but are not limited to, a method of exfoliating the formed sheet-like cell group.
本明細書において、刺激応答性培養基材に被覆されている「刺激応答性高分子」は、例えば、ポリ（Ｎ−イソプロピルアクリルアミド）、ポリ（Ｎ−イソプロピルアクリルアミド−アクリル酸）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−メチルメタクリレート）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−アクリル酸ナトリウム）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−ビニルフェロセン）共重合体、γ線照射したポリ（ビニルメチルエーテル）（ＰＶＭＥ）、ポリ（オキシエチレン）、核酸などの生体物質を高分子に組み込んだ樹脂、及び上記高分子に対して架橋剤によって架橋して作製したゲルなどが挙げられるが、それらに限定されない。 In the present specification, the "stimulation-responsive polymer" coated on the stimulation-responsive culture substrate is, for example, poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-acrylic acid) copolymer, or poly. (N-isopropylacrylamide-methylmethacrylate) copolymer, poly (N-isopropylacrylamide-sodium acrylate) copolymer, poly (N-isopropylacrylamide-vinylferrocene) copolymer, γ-ray irradiated poly (vinylmethyl) Examples thereof include resins in which biological substances such as ether) (PVME), poly (oxyethylene) and nucleic acids are incorporated into polymers, and gels prepared by cross-linking the above polymers with a cross-linking agent, but the present invention is limited thereto. Not done.
本明細書において「温度応答性高分子」は、刺激応答性高分子の１つであり、温度に応答して、その形状及び／又は性質を変化させる高分子をいう。温度応答性高分子としては、例えば、ポリ（Ｎ−イソプロピルアクリルアミド）、ポリ（Ｎ−イソプロピルアクリルアミド−アクリル酸）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−メチルメタクリレート）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−アクリル酸ナトリウム）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−ビニルフェロセン）共重合体、γ線照射したポリ（ビニルメチルエーテル）及び上記高分子に対して架橋剤によって架橋し作製したゲルなどが挙げられるが、それらに限定されない。好ましくは、例えば、ポリ（Ｎ−イソプロピルアクリルアミド）、ポリ（Ｎ−イソプロピルアクリルアミド−メチルメタクリレート）共重合体、ポリ（Ｎ−イソプロピルアクリルアミド−アクリル酸ナトリウム）共重合体及び上記高分子に対して架橋剤によって架橋し作製したゲルなどが挙げられるが、それらに限定されない。 As used herein, the term "temperature-responsive polymer" refers to a polymer that is one of stimulus-responsive polymers and that changes its shape and / or properties in response to temperature. Examples of the temperature-responsive polymer include poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-acrylic acid) copolymer, poly (N-isopropylacrylamide-methylmethacrylate) copolymer, and poly (N-isopropylacrylamide) copolymer. A gel prepared by cross-linking an isopropylacrylamide-sodium acrylate) copolymer, a poly (N-isopropylacrylamide-vinylferrocene) copolymer, a γ-ray irradiated poly (vinyl methyl ether), and the above polymer with a cross-linking agent. Etc., but are not limited to them. Preferably, for example, a cross-linking agent for poly (N-isopropylacrylamide), poly (N-isopropylacrylamide-methylmethacrylate) copolymer, poly (N-isopropylacrylamide-sodium acrylate) copolymer and the above polymer. Examples include, but are not limited to, gels produced by cross-linking with.
本明細書において、培養基材に被覆されている温度応答性高分子としては、例えば、水に対する上限臨界溶液温度（ＵＣＳＴ：Ｕｐｐｅｒ Ｃｒｉｔｉｃａｌ Ｓｏｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ）又は下限臨界溶液温度（ＬＣＳＴ：Ｌｏｗｅｒ Ｃｒｉｔｉｃａｌ Ｓｏｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ）が０〜８０℃であるものが挙げられるがそれらに限定されない。臨界溶液温度とは、高分子の形状及び／又は性質を変化させる閾値の温度をいう。本発明では、好ましくは、ポリ（Ｎ−イソプロピルアクリルアミド）（ＰＩＰＡＡｍ）が被覆された細胞培養基材が使用され得る。 In the present specification, the temperature-responsive polymer coated on the culture substrate is, for example, the upper critical solution temperature (UCST: Upper Critical Solution Temperature) or the lower critical solution temperature (LCST: Lower Critical Solution Temperature) with respect to water. Is 0 to 80 ° C., but is not limited thereto. The critical solution temperature is a threshold temperature at which the shape and / or properties of the polymer are changed. In the present invention, preferably, a cell culture substrate coated with poly (N-isopropylacrylamide) (PIPAAm) can be used.
ポリ（Ｎ−イソプロピルアクリルアミド）（ＰＩＰＡＡｍ）は３２℃に下限臨界溶液温度（ＬＣＳＴ）を有する高分子として知られ、遊離状態であれば、水中で３２℃以上の温度で脱水和を起こしＰＩＰＡＡｍが凝集して白濁する。逆に３２℃未満の温度ではＰＩＰＡＡｍは水和し、水に溶解した状態となる。本発明の一実施態様で用いられる温度応答性培養基材は、ＰＩＰＡＡｍがシャーレやセルカルチャーインサートなどの培養表面に被覆されて固定されたものである。したがって、３２℃以上の温度であれば、培養表面のＰＩＰＡＡｍも同じように脱水和するが、ＰＩＰＡＡｍが培養表面に固定されているために培養表面が疎水性を示す。逆に、３２℃未満の温度では、培養表面のＰＩＰＡＡｍは水和するが、ＰＩＰＡＡｍが培養表面に被覆されているため、培養表面が親水性を示す。疎水性の培養表面は細胞が付着し、増殖することができる表面であり、反対に親水性の培養表面は細胞が付着しにくい表面である。そのため、温度応答性培養基材を３２℃未満に冷却すると、細胞が培養表面から剥離する。細胞が培養表面全体にコンフルエントになるまで培養されていれば、培養表面を３２℃未満に冷却することによってシート状の細胞層（細胞シート）を回収することができる。ＰＩＰＡＡｍが被覆された温度応答性培養基材として、例えば、セルシード社（日本）が製造し、販売しているＵｐＣｅｌｌ（登録商標）を用いることができる。 Poly (N-isopropylacrylamide) (PIPAAm) is known as a polymer having a lower limit critical solution temperature (LCST) at 32 ° C. If it is in a free state, it undergoes dehydration at a temperature of 32 ° C. or higher in water to aggregate PIPAAm. And it becomes cloudy. On the contrary, at a temperature lower than 32 ° C., PIPAAm is hydrated and becomes dissolved in water. The temperature-responsive culture substrate used in one embodiment of the present invention is one in which PIPAAm is coated and fixed on a culture surface such as a petri dish or a cell culture insert. Therefore, at a temperature of 32 ° C. or higher, PIPAAm on the culture surface is similarly dehydrated, but the culture surface is hydrophobic because PIPAAm is fixed to the culture surface. On the contrary, at a temperature of less than 32 ° C., PIPAAm on the culture surface is hydrated, but since PIPAAm is coated on the culture surface, the culture surface is hydrophilic. The hydrophobic culture surface is a surface on which cells can adhere and proliferate, while the hydrophilic culture surface is a surface on which cells are difficult to adhere. Therefore, when the temperature-responsive culture substrate is cooled below 32 ° C., the cells detach from the culture surface. If the cells are cultured until they become confluent on the entire culture surface, the sheet-like cell layer (cell sheet) can be recovered by cooling the culture surface to less than 32 ° C. As the temperature-responsive culture substrate coated with PIPAAm, for example, UpCell (registered trademark) manufactured and sold by CellSeed (Japan) can be used.
本発明の一実施態様において用いられる、刺激応答性培養基材を使用することにより得られる細胞層（細胞シート）は、従来、細胞回収に用いられるディスパーゼ、トリプシンなどのタンパク質分解酵素を使用しないため、細胞層の下面（培養表面と接触していた側の面）が接着性タンパク質を豊富に維持しており、また、細胞−細胞間のデスモゾーム構造も保持しており、複数の細胞層の積層化に適している。 Since the cell layer (cell sheet) obtained by using the stimulus-responsive culture substrate used in one embodiment of the present invention does not use proteolytic enzymes such as dispase and trypsin conventionally used for cell recovery. , The lower surface of the cell layer (the surface on the side that was in contact with the culture surface) maintains abundant adhesive proteins, and also retains the cell-cell desmosome structure, and multiple cell layers are laminated. Suitable for conversion.
本発明の子宮内膜様組織に含まれる第一細胞層または第二細胞層を構成する細胞数は、使用する細胞の状態、種類または動物種などによって異なるために限定することはできないが、例えば、第一細胞層または第二細胞層を形成する時に、０．３×１０4〜１０×１０6個／ｃｍ2であってもよく、０．５×１０4〜８×１０6個／ｃｍ2であってもよく、０．７×１０4〜５×１０6個／ｃｍ2の密度で播種されて形成されたものであってもよい。 The number of cells constituting the first cell layer or the second cell layer contained in the endometrial-like tissue of the present invention cannot be limited because it varies depending on the state, type, animal species, etc. of the cells used, but for example. , 0.3 × 10 4 to 10 × 10 6 pieces / cm 2 when forming the first cell layer or the second cell layer, 0.5 × 10 4 to 8 × 10 6 pieces / cm It may be 2 or it may be formed by sowing at a density of 0.7 × 10 4 to 5 × 10 6 pieces / cm 2 .
一実施態様において、本発明の子宮内膜様組織に含まれる第一細胞層は、１層であってもよく、２層以上（例えば２層、３層、４層、５層またはそれ以上）が積層されたものであってもよい。 In one embodiment, the first cell layer contained in the endometrial tissue of the present invention may be one layer, two or more layers (for example, two layers, three layers, four layers, five layers or more). May be laminated.
一実施態様において、本発明の子宮内膜様組織に含まれる第二細胞層は、１層であってもよく、２層以上（例えば２層、３層、４層、５層またはそれ以上）が積層されたものであってもよい。 In one embodiment, the second cell layer contained in the endometrial tissue of the present invention may be one layer or more (for example, two layers, three layers, four layers, five layers or more). May be laminated.
本発明に用いられる細胞または細胞層（第一細胞層、第二細胞層）は、例えば、特開２０１６−２０４３００号公報に記載の方法を参照することにより、採取および／または培養してもよい。 The cells or cell layers (first cell layer, second cell layer) used in the present invention may be collected and / or cultured, for example, by referring to the method described in JP-A-2016-204300. ..
一実施態様において、本発明の子宮内膜様組織は、さらに第二細胞層の上に受精卵が適用されたものであってもよい。本明細書において、「受精卵」とは、卵生殖を行う生物種の雌雄の配偶子（***と卵子）が結合した細胞、及び、当該細胞の***が進行し、２細胞、４細胞、８細胞、桑実胚を経て胚盤胞に至るまでに含まれる細胞塊（細胞群）をいい、これらの細胞塊も本発明に適用することができる。また、一実施態様において、受精卵を覆う透明帯を開口または除去（ハッチング）した受精卵を本発明に適用してもよい。 In one embodiment, the endometrial-like tissue of the present invention may be one in which a fertilized egg is further applied on the second cell layer. As used herein, the term "fertilized egg" refers to a cell to which male and female gametes (sperm and egg) of a species that reproduce eggs are bound, and the cell division progresses, resulting in 2 cells, 4 cells, 8 cells. It refers to a cell mass (cell group) contained in a cell, a mulberry embryo, and a blastomere, and these cell clusters can also be applied to the present invention. Further, in one embodiment, the fertilized egg in which the zona pellucida covering the fertilized egg is opened or removed (hatched) may be applied to the present invention.
一実施態様において、本発明の子宮内膜様組織に適用され得る受精卵は、１）採卵（卵子を採取）したのちに***と掛け合わせる体外受精、もしくは顕微授精から得られた受精卵；または、２）交配後の任意のほ乳類のメスから採取される受精卵であってもよく、１）または２）により得られる受精卵は、新鮮胚または凍結胚を融解させたものでもよい。また、適用され得る受精卵は、公知の方法によってインビトロで培養して、細胞***を進めた細胞塊（例えば、２細胞、４細胞、８細胞、桑実胚、または胚盤胞）であってもよい。 In one embodiment, the fertilized eggs that can be applied to the endometrial tissue of the present invention are 1) in vitro fertilization (collecting eggs) and then crossing with sperm, or fertilized eggs obtained by microinsemination; 2) The fertilized egg collected from any mammal female after mating may be used, and the fertilized egg obtained by 1) or 2) may be a thawed fresh or frozen embryo. Further, the fertilized egg that can be applied is a cell mass (for example, 2 cells, 4 cells, 8 cells, morula, or blastocyst) that has been cultured in vitro by a known method to promote cell division. May be good.
一実施態様において、本発明の子宮内膜様組織は、例えば、上記に記載の各構成が別々にパッケージングされ、所望の時に子宮内膜様組織を調製することができるキットとして提供されてもよい。 In one embodiment, the endometrial tissue of the present invention may be provided, for example, as a kit in which each of the configurations described above is packaged separately and the endometrial tissue can be prepared at the desired time. Good.
＜＜子宮内膜様組織を製造する方法＞＞
本発明は、
（１）細胞接着性ハイドロゲルを含む支持体の上に、子宮内膜間質細胞を含む第一細胞層と、子宮内膜上皮細胞を含む第二細胞層とを形成する工程であって、ここで前記第二細胞層は、前記第一細胞層の上に形成される、工程；および
（２）前記工程（１）で得られる構築物を培養する工程、
を含む、子宮内膜様組織を製造する方法を提供する。
<< Method for producing endometrial-like tissue >>
The present invention
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Provided is a method for producing endometrial-like tissue, including.
一実施態様において、工程（１）は、
（１−１）細胞接着性ハイドロゲルを含む支持体の上に子宮内膜間質細胞を含む第１細胞層を積層し、前記第１細胞層の上に子宮内膜上皮細胞を含む第二細胞層を積層する工程、であってもよい。
In one embodiment, step (1) is
(1-1) A first cell layer containing endometrial stromal cells is laminated on a support containing cell adhesion hydrogel, and a second layer containing endometrial epithelial cells is laminated on the first cell layer. It may be a step of laminating cell layers.
他の態様において、工程（１）は、
（１−２）前記第一細胞層の上に前記第二細胞層を形成した積層体を、細胞接着性ハイドロゲルを含む支持体の上にのせる工程、であってもよい。細胞接着性ハイドロゲルを含む支持体にのせる前に、あらかじめ、第一細胞層の上に前記第二細胞層を形成した積層体を作製することにより、当該積層体の細胞層間の接着が促進され、積層体のハンドリングが容易となる。また、細胞層を支持体の上にのせる工程を減らすことができ、細菌などのコンタミネーションのリスクを低減することができる。
In another embodiment, step (1) is
(1-2) The step may be a step of placing the laminate having the second cell layer formed on the first cell layer on a support containing a cell-adhesive hydrogel. By preparing a laminate in which the second cell layer is formed on the first cell layer in advance before placing it on a support containing cell-adhesive hydrogel, adhesion between the cell layers of the laminate is promoted. Therefore, the handling of the laminated body becomes easy. In addition, the number of steps of placing the cell layer on the support can be reduced, and the risk of contamination such as bacteria can be reduced.
本発明における第一細胞層および前記第二細胞層を積層する方法については、特に限定されるものではないが、例えば、細胞を細胞培養基材に播種し、その上に細胞外マトリックスタンパクを構成するタンパク（例えば、ラミニン、コラーゲン、カドヘリン、フィブロネクチン、エラスチンまたはビトロネクチン等）および／または代表的な細胞接着性ハイドロゲル素材（例えば、ゼラチン、ヒアルロン酸、キチンまたはキトサン等）を含むゲルを塗布後、さらに細胞を播種して積層体を得る方法；培養細胞をシート状で剥離させ、必要に応じ培養細胞移動治具を用い細胞層を積層化する方法等により得られる。用いられる培養細胞移動治具としては、剥離した細胞層を捕捉し、移動できるものであれば材質、形状共に何ら限定されるものではないが、例えば、膜状、多孔膜状、不織布状または織布状であってもよく、材質としてポリビニリデンジフルオライド（ＰＶＤＦ）、シリコン、ポリエステル、ポリビニルアルコール、ウレタン、セルロース及びその誘導体、キチン、キトサン、コラーゲン、ゼラチン、フィブリングルー等であってもよい。また、用いられる培養細胞移動治具としては、例えば、特開２００７−２２２１５３号公報に記載の装置を用いてもよい。 The method for laminating the first cell layer and the second cell layer in the present invention is not particularly limited, but for example, cells are seeded on a cell culture substrate, and an extracellular matrix protein is formed therein. After applying a gel containing proteins (eg, laminin, collagen, cadoherin, fibronectin, elastin or vitronectin, etc.) and / or typical cell-adhesive hydrogel materials (eg, gelatin, hyaluronic acid, chitin or chitosan, etc.) Further, a method of seeding cells to obtain a laminate; a method of peeling cultured cells in a sheet form and, if necessary, laminating cell layers using a cultured cell transfer jig or the like. The cultured cell transfer jig used is not limited in terms of material and shape as long as it can capture and move the exfoliated cell layer, but for example, it is in the form of a film, a porous film, a non-woven fabric, or a weave. It may be in the form of cloth, and the material may be polyvinylidene fluoride (PVDF), silicon, polyester, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibring loop and the like. Further, as the cultured cell transfer jig to be used, for example, the apparatus described in JP-A-2007-222153 may be used.
一実施形態において、本発明の方法は、前記第一細胞層および前記第二細胞層は、刺激応答性培養基材を用いることにより得られる細胞層であってもよい。これにより、接着性タンパク質を豊富に保持した細胞層が得られ、それによって細胞層と細胞層との間、および細胞層と支持体とがしっかりと接着した子宮内膜様組織が作製される。一実施形態において、前記刺激応答性培養基材は、例えば、温度応答性培養基材であってもよい。 In one embodiment, in the method of the present invention, the first cell layer and the second cell layer may be cell layers obtained by using a stimulus-responsive culture substrate. This results in a cell layer that is rich in adhesive proteins, thereby creating an endometrial-like tissue that is tightly adhered between the cell layers and between the cell layers and the support. In one embodiment, the stimulus-responsive culture substrate may be, for example, a temperature-responsive culture substrate.
一実施態様において、前記工程（１）で得られる構築物を培養する工程により、第一細胞層と、第二細胞層と細胞接着性ハイドロゲルを含む支持体とがしっかりと接着し、受精卵移植後に子宮組織に起こる変化をインビトロにおいて観察可能な子宮内膜様組織を提供可能となる。 In one embodiment, by the step of culturing the construct obtained in the step (1), the first cell layer, the second cell layer and the support containing the cell adhesion hydrogel are firmly adhered to each other, and the fertilized egg is transplanted. It is possible to provide endometrial-like tissue in which changes that occur later in the uterine tissue can be observed in vitro.
一実施形態における本発明の方法は、さらに、（３）前記第二細胞層の上に受精卵を適用する工程、を含んでもよい。 The method of the present invention in one embodiment may further include (3) applying a fertilized egg onto the second cell layer.
＜＜子宮内膜組織モデルを用いた受精卵の着床を促進する因子をスクリーニングする方法＞＞
本発明の子宮内膜様組織、または本発明の方法によって得られる子宮内膜様組織は、受精卵の着床を促進する因子を評価する方法に用いることができる。以下に限定されないが、一実施態様において、本発明の方法は、例えば、
（１）子宮内膜様組織に候補因子を適用する工程；
（２）前記子宮内膜様組織の第二細胞層の上に受精卵を適用し、培養する工程；および、
（３）前記受精卵の着床を評価する工程、
を含んでもよい。
<< Method for screening factors that promote implantation of fertilized eggs using an endometrial tissue model >>
The endometrial-like tissue of the present invention, or the endometrial-like tissue obtained by the method of the present invention, can be used as a method for evaluating factors that promote implantation of fertilized eggs. In one embodiment, the method of the invention is, for example, not limited to:
(1) Step of applying candidate factor to endometrial tissue;
(2) A step of applying and culturing a fertilized egg on the second cell layer of the endometrial tissue;
(3) A step of evaluating the implantation of the fertilized egg,
May include.
一実施態様において、工程（１）で適用する候補因子は、子宮内膜様組織に適用（例えば、培地に添加）してもよく、上記の子宮内膜様組織の製造方法の任意の工程で適応（例えば、培地に添加）してもよい。 In one embodiment, the candidate factor applied in step (1) may be applied to endometrial tissue (eg, added to the medium) in any step of the method for producing endometrial tissue described above. It may be adapted (eg, added to the medium).
一実施形態において、工程（３）の受精卵の着床を評価する方法は、公知の染色方法（以下に限定されないが、例えば、ヘマトキシリン・エオシン（Ｈ＆Ｅ）染色、受精卵および子宮内膜組織を染色する抗体（例えば、抗ＧＡＴＡ３抗体および抗アクチン抗体）を用いた免疫組織化学染色もしくは免疫蛍光染色など）によって、子宮内膜様組織を染色後、任意の顕微鏡を用いることによって、受精卵の着床度合いを評価することができる。それにより、適用した候補因子の効果を評価することができる。また、任意の候補因子の効果を判定するにあたっては、例えば、子宮内膜様組織または受精卵の遺伝子の発現の変化や産生されるタンパク質の解析などの分子生物学的手法などを用いて評価することもできる。 In one embodiment, the method for evaluating implantation of the fertilized egg in step (3) is a known staining method (for example, but not limited to, hematoxylin-eosin (H & E) staining, fertilized egg and endometrial tissue. Implantation of fertilized eggs by staining endometrial-like tissue with immunohistochemical staining or immunofluorescent staining with staining antibodies (eg, anti-GATA3 antibody and anti-actin antibody) and then using any microscope. The floor degree can be evaluated. Thereby, the effect of the applied candidate factor can be evaluated. In determining the effect of any candidate factor, for example, molecular biological techniques such as changes in gene expression in endometrial tissue or fertilized eggs and analysis of produced proteins are used for evaluation. You can also do it.
一実施態様において、候補因子は、例えば、低分子化合物、ペプチド、核酸、タンパク質、哺乳動物（例えば、マウス、ラット、ブタ、ウシ、ヒツジ、サル、ヒトなど）の細胞、組織抽出物又は細胞培養上清、植物由来の化合物又は抽出物（例えば、生薬エキス、生薬由来の化合物）、及び微生物由来の化合物若しくは抽出物又は培養産物などであってもよい。 In one embodiment, the candidate factor is, for example, a low molecular weight compound, peptide, nucleic acid, protein, cell, tissue extract or cell culture of a mammal (eg, mouse, rat, pig, bovine, sheep, monkey, human, etc.). It may be a supernatant, a plant-derived compound or extract (for example, a crude drug extract, a crude drug-derived compound), a microorganism-derived compound or extract, or a culture product.
一実施態様において、例えば、受精卵の着床障害を有する個体由来の細胞を含む、子宮内膜様組織を使用してもよい。 In one embodiment, endometrial-like tissue may be used, for example, which comprises cells from an individual having impaired implantation of a fertilized egg.
以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。なお、本実施例におけるラットを用いた実験プロトコールは、東京女子医科大学の動物実験に関する倫理委員会によって承認されたものであり、米国国立衛生研究所（ＮＩＨ）によって刊行された「実験動物の管理と使用に関する指針」（１９９６年改定版）に沿って実施された。 Hereinafter, the present invention will be described in more detail based on examples, but these are not intended to limit the present invention in any way. The experimental protocol using rats in this example was approved by the Ethics Committee on Animal Experiments at Tokyo Women's Medical University, and was published by the National Institutes of Health (NIH) in "Experimental Animal Management". And guidelines for use ”(revised in 1996).
＜子宮内膜様細胞シートの作製方法＞
３週齢のＳＤラット（系統名：Ｓｌｃ：ＳＤ、日本ＳＬＣ株式会社）を購入し、ＣＯ２にて犠牲死させた。ＳＤラットを開腹し、子宮の両側の卵巣と骨盤をつないでいる靭帯を切離し、子宮広間膜を展開したのち、子宮頸部を切除して子宮を摘出した。その後、摘出した子宮を顕微鏡下で、子宮筋層と子宮内膜に分離した。
<Method of producing endometrial-like cell sheet>
A 3-week-old SD rat (systematic name: Slc: SD, Japan SLC Co., Ltd.) was purchased and killed by CO 2 . The SD rat was laparotomized, the ligaments connecting the ovaries and the pelvis on both sides of the uterus were dissected, the uterine broad membrane was expanded, and then the cervix was resected to remove the uterus. The removed uterus was then separated into the myometrium and endometrium under a microscope.
分離した子宮内膜組織に、０．２５％トリプシン／０．１％ＥＤＴＡ溶液を添加し、３７℃、振盪１５０ｒｐｍで２０分間処理した。その後、５ｍＬのピペッターで７０回〜１００回程度、１０００μＬのピペットマンで２００回程度ピペッティングを行った。同量の１０％ＦＢＳ入り培地を添加し、１００μｍおよび４０μｍのセルストレーナーで濾過した。得られた細胞を１００ｍｍのディッシュに全量播種し、二時間静置した（以下、「細胞選別工程」という。）。 A 0.25% trypsin / 0.1% EDTA solution was added to the separated endometrial tissue and treated at 37 ° C. with shaking at 150 rpm for 20 minutes. Then, pipetting was performed 70 to 100 times with a 5 mL pipettor and about 200 times with a 1000 μL pipetman. Equal amounts of medium with 10% FBS were added and filtered through 100 μm and 40 μm cell strainers. All the obtained cells were seeded in a 100 mm dish and allowed to stand for 2 hours (hereinafter referred to as "cell selection step").
二時間後、上澄みを回収し、ＰＢＳで三回洗浄した。洗浄時のＰＢＳも回収した。この上澄みと洗浄したＰＢＳとを合わせて、主に子宮内膜上皮細胞を含む「子宮内膜上皮細胞懸濁液」を得た。洗浄後にディッシュに接着した細胞を０．２５％トリプシン／０．１％ＥＤＴＡで５分、３７℃で反応させて剥離し、主に子宮内膜間質細胞を含む「子宮内膜間質細胞懸濁液」を得た。 Two hours later, the supernatant was collected and washed three times with PBS. PBS during washing was also collected. The supernatant and the washed PBS were combined to obtain an "endometrial epithelial cell suspension" containing mainly endometrial epithelial cells. After washing, the cells adhering to the dish were reacted with 0.25% trypsin / 0.1% EDTA for 5 minutes at 37 ° C to exfoliate, and "endometrial stromal cell suspension" mainly containing endometrial stromal cells. A turbid liquid was obtained.
子宮内膜上皮細胞を、温度応答性高分子が固定されたセルカルチャーインサートに５．０×１０6／ｄｉｓｈの密度で播種した。３５ｍｍの温度応答性培養皿（ＵｐＣｅｌｌ（登録商標）セルシード社製）に子宮内膜間質細胞を２．５×１０6／ｄｉｓｈの密度で播種した。 Endometrial epithelial cells were seeded in cell culture inserts immobilized with a temperature-responsive polymer at a density of 5.0 × 10 6 / dish. Endometrial stromal cells were seeded in a 35 mm temperature responsive culture dish (UpCell® CellSeed) at a density of 2.5 × 10 6 / dish.
子宮内膜上皮細胞は、以下の組成の培地：
・ＤＭＥＭ／Ｆ１２（１：１）
・ｉｎｓｕｌｉｎ １０μｇ／ｍＬ
・ｔｒａｎｓｆｅｒｒｉｎ １０μｇ／ｍＬ
・ｓｅｌｅｎｉｔｅ ０．０３８μＭ
・ｈｙｄｒｏｃｏｒｔｉｓｏｎｅ １００μｇ／ｍＬ
・ｒｅｔｉｎｏｉｃ ａｃｉｄ ２．５ｎＭ
・Ｌ−ａｓｃｏｒｂｉｃ ａｃｉｄ １００μＭ
・ＥＧＦ １０ｎｇ／ｍＬ
を使用して培養した。
Endometrial epithelial cells are a medium having the following composition:
・ DMEM / F12 (1: 1)
・ Insulin 10 μg / mL
・ Transferrin 10 μg / mL
・ Selenite 0.038 μM
・ Hydrocortisone 100 μg / mL
・ Retinoic acid 2.5nM
・ L-ascorbic acid 100 μM
・ EGF 10 ng / mL
Was cultured using.
子宮内膜間質細胞は、以下の組成の培地：
・ＤＭＥＭ／Ｆ１２（１：１）
・１０％ ＦＢＳ
・１％ Ｐｅｎｉｃｉｌｌｉｎ
・１％ Ｓｔｒｅｐｔｏｍｙｃｉｎ
・１％ Ｈｅｐｅｓ
を使用して培養した。
Endometrial stromal cells have the following composition:
・ DMEM / F12 (1: 1)
・ 10% FBS
・ 1% Penicillin
・ 1% Streptomycin
・ 1% Hepes
Was cultured using.
播種４日後に、子宮内膜上皮細胞および子宮内膜間質細胞を培養している培養基材をそれぞれ２０℃で１時間インキュベートし、細胞シートを得た（以下、子宮内膜上皮細胞懸濁液より得られた細胞シートを「上皮細胞シート」、子宮内膜間質細胞懸濁液より得られた細胞シートを「間質細胞シート」という。）。剥離した細胞シートは培地またはＰＢＳに浮遊させた。 Four days after seeding, the culture substrate in which the endometrial epithelial cells and endometrial stromal cells were cultured was incubated at 20 ° C. for 1 hour, respectively, to obtain cell sheets (hereinafter, endometrial epithelial cell suspension). The cell sheet obtained from the fluid is referred to as "epithelial cell sheet", and the cell sheet obtained from the endometrial stromal cell suspension is referred to as "stromal cell sheet"). The exfoliated cell sheet was suspended in medium or PBS.
上記で作製した間質細胞シート２層重ね、その上に上皮細胞シート１層を積層し、合計３層の積層化細胞シート（以下、「子宮内膜様細胞シート」という。）を得た（図１ａおよび図２参照）。 Two layers of the stromal cell sheet prepared above were laminated, and one layer of the epithelial cell sheet was laminated on the layer to obtain a total of three layers of laminated cell sheets (hereinafter referred to as "endometrium-like cell sheets") (hereinafter referred to as "endometrial-like cell sheets"). 1a and 2).
＜比較例１＞
網目構造物であるセルカルチャーインサートが、上記の手順により作製した子宮内膜様細胞シートを接着させる足場として使用可能か検証を行った。
<Comparative example 1>
It was verified whether the cell culture insert, which is a network structure, can be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.
ＯＨＰフィルムを用いて、子宮内膜様細胞シートを、ポアサイズ０．４μｍ、１．０μｍ、３．０μｍまたは８．０μｍのセルカルチャーインサート（ＦＡＬＣＯＮ、＃３５３４９３、＃３５３１０２、＃３５３０９２、＃３５３０９３）の上に移動させて、１．５時間、静置した。 Using transparencies, endometrial cell sheets of cell culture inserts with pore sizes of 0.4 μm, 1.0 μm, 3.0 μm or 8.0 μm (FALCON, # 353493, # 353102, # 353092, # 353093) It was moved up and allowed to stand for 1.5 hours.
セルカルチャーインサートに生着させた子宮内膜様細胞シートを、１％ Ｐｅｎｉｃｉｌｌｉｎ及びＳｔｒｅｐｔｏｍｙｃｉｎ含有のＤＭＥＭ／Ｆ１２培地（ナカライテスク、＃０５１７７−１５）に浸漬させ、３７℃、５％ＣＯ２にて３日間培養した。セルカルチャーインサート上で培養した子宮内膜様細胞シートは、ポアサイズ１．０μｍ、３．０μｍおよび８．０μｍセルカルチャーインサート上といずれの場合においても３日後には完全に剥離して収縮した（図３）。ポアサイズ０．４μｍのセルカルチャーインサート（ＦＡＬＣＯＮ社、＃３５３４９３）も同様に、３日後には完全に剥離して収縮した（図示せず）。 The endometrial-like cell sheet engrafted in the cell culture insert was immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Penicillin and Streptomycin, and 3 at 37 ° C. and 5% CO 2 . Incubated for days. Endometrial cell sheets cultured on cell culture inserts were completely exfoliated and contracted after 3 days on pore sizes 1.0 μm, 3.0 μm and 8.0 μm cell culture inserts (Fig.). 3). Similarly, a cell culture insert having a pore size of 0.4 μm (FALCON, # 353493) completely peeled off and contracted after 3 days (not shown).
＜実施例１＞
コラーゲンゲルが、上記の手順により作製した子宮内膜様細胞シートを接着させる足場として使用可能か検証を行った。
<Example 1>
It was verified whether the collagen gel can be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.
コラーゲンゲルは、以下：
・２．４ｍＬの５ｍｇ／ｍＬ コラーゲン酸性溶液Ｉ−ＡＣ（ＫＯＫＥＮ、＃ＩＡＣ−５０）
・０．３ｍＬの１０倍濃縮培地（Ｇｉｂｃｏ）
・０．０３ｍＬの１０００ｍＭ ＮａＨＣＯ３（和光純薬、＃１９１−０１３０５）
・０．０３ｍＬの１０００ｍＭ Ｈｅｐｅｓ（Ｓｉｇｍａ−ａｌｄｒｉｃｈ、＃Ｈ３３７５−５００Ｇ）
・０．２４ｍＬの蒸留水
を混合し、Ｓａｋａｇｕｃｈｉら（国際公開第２０１２／０３６２２５）に記載のアクリルフレームに流し込み、硬化させることで作製した。
Collagen gel is as follows:
-2.4 mL of 5 mg / mL collagen acidic solution I-AC (KOKEN, # IAC-50)
-0.3 mL 10-fold concentrated medium (Gibco)
-0.03 mL of 1000 mM NaHCO 3 (Wako Pure Drug, # 191-01305)
0.03 mL of 1000 mM Hepes (Sigma-aldrich, # H3375-500G)
-It was prepared by mixing 0.24 mL of distilled water, pouring it into an acrylic frame described in Sakaguchi et al. (International Publication No. 2012/036225), and curing it.
子宮内膜様細胞シートをＯＨＰフィルムに載せて、作製したコラーゲンゲル上へ移動させ、１．５時間、静置した。 The endometrial-like cell sheet was placed on an OHP film, transferred onto the prepared collagen gel, and allowed to stand for 1.5 hours.
コラーゲンゲルに生着させた子宮内膜様細胞シートを、１％ Ｐｅｎｉｃｉｌｌｉｎ及びＳｔｒｅｐｔｏｍｙｃｉｎ含有のＤＭＥＭ／Ｆ１２培地（ナカライテスク社、＃０５１７７−１５）に浸漬させ、３７℃、５％ＣＯ２にて３日間培養した。その結果、細胞シートはコラーゲンゲル上に生着したが、一部は剥離した（図４ｂ）。ＨＥ染色を行い、コラーゲンゲル上に接着させた細胞シートを観察した結果、細胞シートとゲルとの間に隙間が観察された（図４ｃ）。 The endometrial-like cell sheet engrafted in the collagen gel was immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Penicillin and Streptomycin, and 3 at 37 ° C. and 5% CO 2 . Incubated for days. As a result, the cell sheet engrafted on the collagen gel, but part of it was exfoliated (Fig. 4b). As a result of performing HE staining and observing the cell sheet adhered on the collagen gel, a gap was observed between the cell sheet and the gel (Fig. 4c).
＜実施例２＞
次に、コラーゲンゲルとフィブリンゲルを混合して作製した足場が、上記の手順により作製した子宮内膜様細胞シートを接着させる足場として使用可能か検証を行った。
<Example 2>
Next, it was verified whether the scaffold prepared by mixing collagen gel and fibrin gel could be used as a scaffold for adhering the endometrial-like cell sheet prepared by the above procedure.
フィブリンは血液の凝固に関わるタンパク質であり、細胞接着性を有する。フィブリノゲンにトロンビンが反応することによりフィブリンへと変化し、フィブリン同士が連鎖的に反応することによりフィブリン膜を形成し、組織のすき間を埋める。フィブリン膜は、生体中に存在するプラスミンという酵素によって分解されるため、この酵素の働きを止めるアプロチニンを加える。細胞シートを接着させる足場に使用する場合も、フィブリノゲン、トロンビンおよびアプロチニンの３つを混合させてフィブリンを作製する。コラーゲンはフィブリノゲン、トロンビン、アプロチニンを支える土台となり、マイクロチャネルを有するゲルをつくることが可能となる。 Fibrin is a protein involved in blood coagulation and has cell adhesion. Thrombin reacts with fibrinogen to change into fibrin, and fibrin reacts in a chain reaction to form a fibrin membrane, filling the gaps in tissues. Since the fibrin membrane is decomposed by an enzyme called plasmin that exists in the living body, aprotinin that stops the action of this enzyme is added. When used as a scaffold for adhering cell sheets, fibrinogen, thrombin and aprotinin are mixed to prepare fibrin. Collagen provides the basis for supporting fibrinogen, thrombin, and aprotinin, making it possible to create gels with microchannels.
フィブリンゲル／コラーゲンゲルは、以下：
・１．５ｍＬの２５ｍｇ／ｍＬ フィブリノゲン（Ｓｉｇｍａ−ａｌｄｒｉｃｈ、＃Ｆ８６３０−５Ｇ）
・１．５ｍＬの２．５Ｕ／ｍＬ トロンビン（Ｓｉｇｍａ−ａｌｄｒｉｃｈ、＃Ｔ４６４８−１ＫＵ）
・０．１５ｍＬの７５０００ＫＩＵ／ｍＬ アプロチニン（和光純薬社、＃０１６−１１８３６）
・２．４ｍＬの５ｍｇ／ｍＬ コラーゲン酸性溶液Ｉ−ＡＣ（ＫＯＫＥＮ 社、＃ＩＡＣ−５０）
・０．３ｍＬの１０倍濃縮培地（Ｇｉｂｃｏ）
・０．０３ｍＬの１０００ｍＭ ＮａＨＣＯ３（和光純薬、＃１９１−０１３０５）
・０．０３ｍＬの１０００ｍＭ Ｈｅｐｅｓ（Ｓｉｇｍａ−ａｌｄｒｉｃｈ、＃Ｈ３３７５−５００Ｇ）
・０．２４ｍＬの蒸留水
を混合し、Ｓａｋａｇｕｃｈｉら（国際公開第２０１２／０３６２２５）に記載のアクリルフレームに流し込み、硬化させて作製した。
Fibrin gel / collagen gel is as follows:
1.5 mL 25 mg / mL fibrinogen (Sigma-aldrich, # F8630-5G)
-1.5 mL of 2.5 U / mL thrombin (Sigma-aldrich, # T4648-1KU)
0.15 mL 75000 KIU / mL aprotinin (Wako Junyakusha, # 016-11836)
-2.4 mL of 5 mg / mL collagen acidic solution I-AC (KOKEN, # IAC-50)
-0.3 mL 10-fold concentrated medium (Gibco)
-0.03 mL of 1000 mM NaHCO 3 (Wako Pure Drug, # 191-01305)
0.03 mL of 1000 mM Hepes (Sigma-aldrich, # H3375-500G)
-0.24 mL of distilled water was mixed, poured into an acrylic frame described by Sakaguchi et al. (International Publication No. 2012/036225), and cured to prepare a product.
子宮内膜様細胞シートをＯＨＰフィルムにのせて、フィブリンゲル／コラーゲンゲル上へ移動させ、１．５時間静置した。 The endometrial-like cell sheet was placed on an OHP film, transferred onto a fibrin gel / collagen gel, and allowed to stand for 1.5 hours.
フィブリンゲル／コラーゲンゲルに生着させた子宮内膜様細胞シートを、１％ Ｐｅｎｉｃｉｌｌｉｎ及びＳｔｒｅｐｔｏｍｙｃｉｎ含有のＤＭＥＭ／Ｆ１２培地（ナカライテスク、＃０５１７７−１５）に浸漬させ、３７℃、５％ＣＯ２にて３日間培養した。その結果、フィブリンゲル／コラーゲンゲル上で培養した子宮内膜様細胞シートは３日後でも剥離せず、移植直後と同等の大きさの子宮内膜様細胞シートが観察された（図５）。ＨＥ染色を行い、フィブリンゲル／コラーゲンゲル上に接着させた子宮内膜様細胞シートを観察した結果、子宮内膜様細胞シートとゲルの間に隙間はなく、上皮層と間質層の二層構造であることが確認された（図６ａ）。この構造は正常な子宮内膜構造と類似していた。 Endometrial cell sheets engrafted in fibrin gel / collagen gel are immersed in DMEM / F12 medium (Nacalai Tesque, # 05177-15) containing 1% Pencillin and Streptomycin, and brought to 37 ° C. and 5% CO 2 . Was cultured for 3 days. As a result, the endometrial-like cell sheet cultured on the fibrin gel / collagen gel did not peel off even after 3 days, and an endometrial-like cell sheet having the same size as immediately after transplantation was observed (Fig. 5). As a result of performing HE staining and observing the endometrial-like cell sheet adhered on the fibrin gel / collagen gel, there was no gap between the endometrial-like cell sheet and the gel, and there were two layers, an epithelial layer and an stromal layer. The structure was confirmed (Fig. 6a). This structure was similar to the normal endometrial structure.
子宮内膜様細胞シートを端から１ｍｍ間隔で厚みを測定したところ、平均５１．７μｍの厚みであった（図６ｂ）。 When the thickness of the endometrial-like cell sheet was measured at 1 mm intervals from the edge, the average thickness was 51.7 μm (FIG. 6b).
子宮内膜様細胞シートの組織切片を、ＤＡＰＩ（青）、抗ＣＫ１８抗体（緑）（Ａｂｃａｍ社、＃ａｂ５３１１８）、抗ビメンチン抗体（赤）（Ａｂｃａｍ社、＃ａｂ２０３４６）を用いて免疫染色し、蛍光顕微鏡で観察した。その結果、最上層は上皮マーカーであるＣＫ１８陽性であることが確認された（図７ａ）。また、その直下には、間質マーカーであるビメンチン陽性の層が確認された。 Tissue sections of the endometrial cell sheet were immunostained with DAPI (blue), anti-CK18 antibody (green) (Abcam, # ab53118), anti-vimentin antibody (red) (Abcam, # ab20346). It was observed with a fluorescence microscope. As a result, it was confirmed that the uppermost layer was positive for the epithelial marker CK18 (Fig. 7a). In addition, a layer positive for vimentin, which is an interstitial marker, was confirmed immediately below it.
子宮内膜様細胞シートの組織切片を、ＤＡＰＩ（青）、抗エストロゲンレセプター抗体（緑）（Ａｂｃａｍ社、＃ａｂ３５７７）、抗プロゲステロン抗体（赤）（Ａｂｃａｍ社、＃ａｂ２７６４）を用いて免疫染色し、蛍光顕微鏡で観察した。その結果、図７ｂに示すようにエストロゲンレセプター陽性、及びプロゲステロン陽性の層が確認された。 Tissue sections of the endometrial cell sheet were immunostained with DAPI (blue), anti-estrogen receptor antibody (green) (Abcam, # ab3577), and anti-progesterone antibody (red) (Abcam, # ab2764). , Observed with a fluorescence microscope. As a result, as shown in FIG. 7b, an estrogen receptor positive and a progesterone positive layer were confirmed.
以上の結果から、フィブリンゲル／コラーゲンゲルからなる足場は、インビトロにおいて、子宮内膜様細胞シートを安定的に維持できることが明らかとなった。 From the above results, it was clarified that the scaffold made of fibrin gel / collagen gel can stably maintain the endometrial-like cell sheet in vitro.
＜実施例３＞
子宮内膜様組織を用いた受精卵の移植評価
<Example 3>
Evaluation of transplantation of fertilized eggs using endometrial-like tissue
受精卵は分割を繰り返し、二細胞を経て、およそ３日で胚盤胞に達する。ラットにおいて、受精卵は胚盤胞の状態で着床する。本発明の子宮内膜様組織が、生体と同様に受精卵の着床が再現可能か検証を行った。 The fertilized egg repeats division, passes through two cells, and reaches the blastocyst in about 3 days. In rats, fertilized eggs implant in the state of blastocysts. It was verified whether the endometrial tissue of the present invention can reproduce the implantation of a fertilized egg in the same manner as the living body.
市販の凍結受精卵（アークリソース社）を解凍し、受精卵用培地（ｍＲ１ＥＣＭ（アークリソース社）で１日間培養して胚盤胞を形成させた。得られた胚盤胞は、グレード分類において４ＡＡであり、良好胚であった。 Commercially available frozen fertilized eggs (Arc Resources) were thawed and cultured in a fertilized egg medium (mR1ECM (Arc Resources) for 1 day to form blastocysts. The obtained blastocysts were classified according to grade. It was 4AA and was a good embryo.
透明帯からハッチングした胚盤胞を、実施例２と同様の方法により得られたフィブリンゲル／コラーゲンゲル＋子宮内膜様細胞シート（以下、「子宮内膜様組織」という。）の上に載せ、受精卵用培地に浸漬させた状態で、３７℃、５％ＣＯ２にて３６時間培養した。 The blastocyst hatched from the zona pellucida is placed on a fibrin gel / collagen gel + endometrial cell sheet (hereinafter referred to as "endometrial tissue") obtained by the same method as in Example 2. , Immersed in fertilized egg medium, cultured at 37 ° C. and 5% CO 2 for 36 hours.
その後、子宮内膜様組織を抗ＧＡＴＡ３抗体（赤）（Ａｂｃａｍ、＃ａｂ１０６６２５）と、抗アクチン抗体（緑）（ｉｎｖｉｔｒｏｇｅｎ、＃１９４１１２２）を用いて免疫染色し、共焦点レーザー顕微鏡（ＦＶ３０００；オリンパス）で観察した。 The endometrial tissue was then immunostained with an anti-GATA3 antibody (red) (Abcam, # ab106625) and an anti-actin antibody (green) (invitrogen, # 1941122) and confocal laser scanning microscope (FV3000; Olympus). Observed at.
その結果、ＧＡＴＡ３陽性部位（すなわち、受精卵）がアクチン陽性部位（すなわち、子宮内膜組織）に囲まれている様子が観察された。これは、受精卵（胚盤胞）が着床した際に起こる初期の浸潤過程を模倣しているものと考えられる。この実験系を用いて、初期の浸潤過程での受精卵・上皮・間質組織の関係性を解明できる可能性が示唆された。 As a result, it was observed that the GATA3 positive site (that is, the fertilized egg) was surrounded by the actin positive site (that is, the endometrial tissue). This is thought to mimic the initial infiltration process that occurs when a fertilized egg (blastocyst) implants. It was suggested that this experimental system could be used to elucidate the relationship between fertilized eggs, epithelium, and stromal tissue during the initial infiltration process.
本明細書に引用する全ての刊行物及び特許文献は、参照により全体として本明細書中に援用される。なお、例示を目的として、本発明の特定の実施形態を本明細書において説明したが、本発明の精神及び範囲から逸脱することなく、種々の改変が行われる場合があることは、当業者に容易に理解されるであろう。 All publications and patent documents cited herein are incorporated herein by reference in their entirety. Although specific embodiments of the present invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.
Claims (18)
前記支持体の上に形成された、子宮内膜間質細胞を含む第一細胞層と、
前記第一細胞層の上に形成された、子宮内膜上皮細胞を含む第二細胞層と
を含む、子宮内膜様組織。 With a support containing cell adhesion hydrogel,
A first cell layer containing endometrial stromal cells formed on the support,
Endometrial-like tissue formed on the first cell layer and comprising a second cell layer containing endometrial epithelial cells.
（１）細胞接着性ハイドロゲルを含む支持体の上に、子宮内膜間質細胞を含む第一細胞層と、子宮内膜上皮細胞を含む第二細胞層とを形成する工程であって、ここで前記第二細胞層は、前記第一細胞層の上に形成される、工程；および
（２）前記工程（１）で得られる構築物を培養する工程、
を含む、方法。 A method for producing endometrial-like tissue, such as:
(1) A step of forming a first cell layer containing endometrial stromal cells and a second cell layer containing endometrial epithelial cells on a support containing cell-adhesive hydrogel. Here, the second cell layer is formed on the first cell layer; and (2) the step of culturing the structure obtained in the step (1).
Including methods.
前記第一細胞層の上に前記第二細胞層を形成した積層体を、前記支持体の上にのせる工程である、請求項８〜１１のいずれか１項に記載の方法。 The step (1)
The method according to any one of claims 8 to 11, which is a step of placing the laminate having the second cell layer formed on the first cell layer on the support.
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| WO2023021591A1 (en) * | 2021-08-18 | 2023-02-23 | 国立大学法人東北大学 | Endometrium model, method for producing endometrium model, and endometrial implantation model |
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| CN112941012B (en) * | 2021-02-09 | 2023-03-31 | 华南农业大学 | Preparation method and application of purified uterine cavity epithelial cells, glandular epithelial cells and stromal cells |
| WO2023021591A1 (en) * | 2021-08-18 | 2023-02-23 | 国立大学法人東北大学 | Endometrium model, method for producing endometrium model, and endometrial implantation model |
| WO2023182287A1 (en) * | 2022-03-23 | 2023-09-28 | 国立大学法人九州大学 | Embryo culture method |
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