JP2015082987A - Method for activating sperm, and application of the same - Google Patents
Method for activating sperm, and application of the same Download PDFInfo
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- JP2015082987A JP2015082987A JP2013222630A JP2013222630A JP2015082987A JP 2015082987 A JP2015082987 A JP 2015082987A JP 2013222630 A JP2013222630 A JP 2013222630A JP 2013222630 A JP2013222630 A JP 2013222630A JP 2015082987 A JP2015082987 A JP 2015082987A
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Abstract
Description
本発明は再生医療に有用な技術に関する。詳しくは、精子の活性化方法及びその用途に関する。 The present invention relates to a technique useful for regenerative medicine. Specifically, it relates to a method for activating sperm and its use.
ヒト人工多能性幹（iPS）細胞の登場によって、再生医療の実現や適用拡大に向けた研究が加速している。iPS細胞はES細胞に比べて倫理的な問題が少なく、また、様々な症例に適用できる可能性を秘める。一方、安全性や入手の容易性等の点から、体細胞、特に組織幹細胞（体性幹細胞）に対する期待も依然として大きい。例えば、多能性幹細胞の一つである間葉系幹細胞（MSCs）は骨細胞、軟骨細胞、心筋細胞など、様々な細胞に分化する能力を有し、その臨床応用に注目が集まっている。一方、多能性幹細胞源として脂肪組織が有望であることがいくつかの研究グループによって報告されている（非特許文献１）。また、北川らによって、脂肪組織より、多能性を示す細胞集団を簡便な操作で大量に調製することが可能であることが報告されるとともに、得られた細胞が脂肪組織への分化能を有し、脂肪組織の再建に有効であることが示された（特許文献１）。本発明者らの研究グループも脂肪組織由来幹細胞（Adipose-derived stem cells: ASC、Adipose-derived regeneration cells: ADRC、Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSCなどと呼ばれる）が様々な疾患に有効であることを報告した（特許文献２〜４）。 With the advent of human induced pluripotent stem (iPS) cells, research aimed at realizing regenerative medicine and expanding its application is accelerating. iPS cells have fewer ethical problems than ES cells, and have the potential to be applied to various cases. On the other hand, expectations for somatic cells, particularly tissue stem cells (somatic stem cells), are still high from the viewpoint of safety and availability. For example, mesenchymal stem cells (MSCs), which are one of pluripotent stem cells, have the ability to differentiate into various cells such as bone cells, chondrocytes and cardiomyocytes, and their clinical application is attracting attention. On the other hand, some research groups have reported that adipose tissue is promising as a source of pluripotent stem cells (Non-patent Document 1). Kitagawa et al. Reported that it is possible to prepare a large number of cell populations showing pluripotency from adipose tissue by a simple operation, and that the obtained cells have the ability to differentiate into adipose tissue. It has been shown to be effective for the reconstruction of adipose tissue (Patent Document 1). Our research group also has various adipose tissue-derived stem cells (Adipose-derived stem cells: ASC, Adipose-derived regeneration cells: ADRC, Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSC, etc.) It was reported to be effective for diseases (Patent Documents 2 to 4).
現代社会において不妊症に悩む夫婦は多く、その対処法（治療法）に対するニーズは大きい。不妊症の中でも、主たる原因が男性側に認められるものは男性不妊症と呼ばれる。男性不妊症の一つとして、陰嚢静脈逆流または鬱滞により精巣温度が上昇し、精子運動率が低下する精索静脈瘤の病態がある。また、獣医学では夏の室内温度上昇のために上記同様な病態で、いわゆる夏季不妊症と呼ばれるブタ精子の運動率低下が毎年夏季に発生し、人工授精用精液の供給に不安定性を来している。このような背景の下で本発明は、体細胞（特に組織幹細胞）の新規な用途として、男性不妊症の治療ないし改善に有効な手段、畜産や育種の分野における人工授精の成功率や効率を向上させる手段などを提供することを目的とする。 There are many couples who suffer from infertility in the modern society, and there is a great need for coping methods (treatment methods). Among infertility, those whose main cause is recognized by men are called male infertility. One type of male infertility is the pathology of varicocele in which testicular temperature rises due to scrotal vein reflux or stagnation and sperm motility decreases. In addition, in veterinary medicine, due to a rise in indoor room temperature in summer, a decrease in the motility rate of porcine sperm, so-called summer infertility, occurs in summer every year, causing instability in the supply of semen for artificial insemination. ing. Under such a background, the present invention provides a novel use of somatic cells (especially tissue stem cells) as an effective means for the treatment or improvement of male infertility, and the success rate and efficiency of artificial insemination in the fields of livestock and breeding. It aims at providing the means etc. which improve.
本発明者らのこれまでの経験から、間葉系幹細胞シート上での培養が幹細胞の増殖に有効であることが判明している。この知見を考慮しつつ、組織幹細胞の一つである脂肪組織由来幹細胞（以下、「ASC」と呼ぶことがある）の潜在能力に注目し、精子に対する脂肪組織由来幹細胞の効果・影響を調べることにした。具体的には、ヒト精索静脈瘤において精子運動性の低下により発生する男子不妊症を治療するための新規細胞療法を開発する目的の下、本疾患と同一の病態を持つブタ夏季不妊症の精子とブタ脂肪組織由来幹細胞を用い、共培養による治療を試みた。また、準備実験として、牛精子とブタ脂肪組織由来幹細胞との共培養を試みた。更には、ブタ精子に及ぼすヒト幹細胞の治療効果を調べた。詳細な検討の結果、（１）脂肪組織由来幹細胞は共培養により精子の運動率を高く維持し、且つ精子の前進運動性を活性化又は高率に維持すること、（２）脂肪組織由来幹細胞の精子活性化作用は、分泌されるサイトカインが介在するのではなく、精子と脂肪組織由来幹細胞の接触によること、及び（３）精子と脂肪組織由来幹細胞の共培養による効果（精子活性化）は種を超えて認められること、が明らかとなり、結論として、この現象はヒト静脈瘤による男子不妊症の細胞治療として応用可能であり、精子運動率低下の病態において種を超えて有用な治療技術と考えられた。一方、大型動物（ブタ）を用いて体外受精実験を行ったところ、脂肪組織由来幹細胞が精子の運動性を劇的に改善し、受精率を向上させることが明らかとなった。更なる検討として、脂肪組織由来幹細胞以外の体細胞の有効性を調べたところ、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞も精子を活性化することが判明した。また、細胞が産生する液性の因子によるのではなく、細胞と精子の接触によって精子が活性化することが裏づけられた。
以下の発明は主として以上の成果に基づく。
［１］以下のステップ（１）を含む、精子活性化方法：
（１）脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞と、精子とを共培養するステップ。
［２］前記細胞と前記精子が接触した状態で共培養される、［１］に記載の方法。
［３］ステップ（１）において、前記細胞が細胞層を形成しており、該細胞層の上で前記精子が培養される、［１］又は［２］に記載の方法。
［４］以下のステップ（２）を更に含む、［１］〜［３］のいずれか一項に記載の方法：
（２）共培養後の精子を回収するステップ。
［５］ステップ（２）において、共培養後の精子が共培養後の前記細胞から分離した状態で回収される、［４］に記載の方法。
［６］前記細胞として、脂肪組織由来幹細胞が用いられる、［１］〜［５］のいずれか一項に記載の方法。
［７］前記細胞の生物種と前記精子の生物種が同一である、［１］〜［６］のいずれか一項に記載の方法。
［８］前記精子がヒト精子である、［１］〜［７］のいずれか一項に記載の方法。
［９］前記精子が非ヒト哺乳動物精子である、［１］〜［７］のいずれか一項に記載の方法。
［１０］前記非ヒト哺乳動物がブタ、ウシ、ウマ、ヤギ又はヒツジである、［９］に記載の方法。
［１１］［１］〜［１０］のいずれか一項に記載の方法で活性化した精子。
［１２］［１１］に記載の精子を含有する、人工授精用精子組成物。
［１３］脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞と精子を含有する、人工授精用精子組成物。
［１４］脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞を含有する精子活性化剤。
［１５］脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞とともに精子を非ヒト哺乳動物の子宮に注入することを特徴とする、人工授精方法。
［１６］脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞の存在下、生体外で卵子と精子を共存させることを特徴とする、体外受精方法。
Our previous experience has revealed that culturing on mesenchymal stem cell sheets is effective for stem cell proliferation. Considering this knowledge, we will focus on the potential of adipose tissue-derived stem cells (hereinafter sometimes referred to as “ASC”), one of the tissue stem cells, and investigate the effects and influence of adipose tissue-derived stem cells on sperm. I made it. Specifically, for the purpose of developing a new cell therapy to treat male infertility that occurs due to decreased sperm motility in human varicocele, a case of swine summer infertility with the same pathology as this disease was developed. Using sperm and porcine adipose tissue-derived stem cells, we tried treatment by co-culture. As a preparatory experiment, co-culture of bovine sperm and porcine adipose tissue-derived stem cells was attempted. Furthermore, the therapeutic effect of human stem cells on porcine sperm was examined. As a result of detailed studies, (1) adipose tissue-derived stem cells maintain high sperm motility by co-culture and activate or maintain high sperm forward motility, (2) adipose tissue-derived stem cells The sperm activation action is not mediated by secreted cytokines, but is due to contact between sperm and adipose tissue-derived stem cells, and (3) the effect of culturing sperm and adipose tissue-derived stem cells (sperm activation) As a conclusion, this phenomenon can be applied as a cell therapy for male infertility due to human varicose veins. it was thought. On the other hand, in vitro fertilization experiments using large animals (pigs) revealed that adipose tissue-derived stem cells dramatically improved sperm motility and increased fertilization rate. As a further study, when the effectiveness of somatic cells other than adipose tissue-derived stem cells was examined, it was found that bone marrow-derived stem cells, dental pulp-derived stem cells and fibroblasts also activate sperm. Moreover, it was confirmed that the sperm was activated by the contact of the cell and the sperm, not by the humoral factor produced by the cell.
The following invention is mainly based on the above results.
[1] A sperm activation method comprising the following step (1):
(1) A step of co-culturing a sperm with a cell selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells and fibroblasts.
[2] The method according to [1], wherein the cells and the sperm are co-cultured in contact with each other.
[3] The method according to [1] or [2], wherein, in step (1), the cells form a cell layer, and the sperm is cultured on the cell layer.
[4] The method according to any one of [1] to [3], further comprising the following step (2):
(2) A step of collecting sperm after co-culture.
[5] The method according to [4], wherein in step (2), the sperm after co-culture is recovered in a state separated from the cells after co-culture.
[6] The method according to any one of [1] to [5], wherein adipose tissue-derived stem cells are used as the cells.
[7] The method according to any one of [1] to [6], wherein the biological species of the cell and the biological species of the sperm are the same.
[8] The method according to any one of [1] to [7], wherein the sperm is a human sperm.
[9] The method according to any one of [1] to [7], wherein the sperm is a non-human mammal sperm.
[10] The method according to [9], wherein the non-human mammal is a pig, cow, horse, goat or sheep.
[11] A sperm activated by the method according to any one of [1] to [10].
[12] A sperm composition for artificial insemination, comprising the sperm according to [11].
[13] A sperm composition for artificial insemination, comprising cells and sperm selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells, and fibroblasts.
[14] A sperm activator comprising a cell selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells, and fibroblasts.
[15] An artificial insemination method characterized by injecting sperm into a uterus of a non-human mammal together with cells selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells and fibroblasts.
[16] An in vitro fertilization method comprising coexisting an ovum and a sperm in vitro in the presence of a cell selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells, and fibroblasts.
１．精子活性化方法
本発明の第１の局面は精子活性化方法（以下、「本発明の方法」とも呼ぶ）に関する。本発明の方法によれば活性化された精子が得られる。従って、本発明の方法は、活性化精子の生産ないし取得方法ともいえる。精子について使用する用語「活性化」とは、精子の運動性／運動率（特に前進運動性／前進運動率）が高まることを意味する。運動性／運動率の低下を抑え、高い運動性／運動率を維持することも、ここでの「運動性／運動率が高まること」に該当する。精子の活性化は、卵子突入率の向上及び／又は受精率の向上をもたらす。
1. Sperm Activation Method The first aspect of the present invention relates to a sperm activation method (hereinafter also referred to as “method of the present invention”). According to the method of the present invention, activated sperm is obtained. Therefore, it can be said that the method of the present invention is a method for producing or obtaining activated sperm. The term “activation” as used for sperm means an increase in sperm motility / motility (particularly forward motility / forward motility). The suppression of the decrease in the motility / motility rate and the maintenance of the high motility / motility rate also correspond to the “increase in the motility / motility rate” here. The activation of sperm results in an increase in egg entry rate and / or an increase in fertilization rate.
本発明の方法では、特定の細胞と精子とを共培養する（ステップ（１））。当該ステップは、その定義から明らかな通り、in vitro（生体外）で実施される。ここでの特定の細胞（説明の便宜上、以下では「共培養用細胞」とも呼ぶ）として、脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞が用いられる。典型的にはこれらの細胞のいずれか、即ち一種類の細胞が用いられるが、二種以上の細胞を併用することを妨げるものではない。後述の実施に示すように、脂肪組織由来幹細胞に最も高い精子活性化効果を認めた。この知見に基づき、好ましくは、共培養用細胞として脂肪組織由来幹細胞を用いる。 In the method of the present invention, specific cells and sperm are co-cultured (step (1)). This step is performed in vitro (in vitro), as is clear from its definition. As specific cells here (for convenience of explanation, also referred to as “cells for co-culture” below), adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells or fibroblasts are used. Typically, one of these cells, ie one type of cell, is used, but this does not preclude the use of two or more types of cells in combination. As shown in the implementation described later, the highest sperm activation effect was observed in the adipose tissue-derived stem cells. Based on this finding, preferably, adipose tissue-derived stem cells are used as co-culture cells.
細胞の由来、即ち生物種は特に限定されないが、本発明の方法に使用する精子の由来、及び本発明の方法によって得られる精子（活性化された精子）の用途を考慮して細胞の由来を決定するとよい。例えば、精子の由来がヒトであり、活性化された精子の用途がヒトの疾患の治療であれば、好ましくはヒト細胞を精子との共培養に供する。このように、好ましくは、共培養細胞の生物種と精子の生物種を同一にする。但し、特定の細胞と精子との共培養による所望の効果（即ち、精子の活性化）が種を超えて認められるという驚くべき知見（詳細は後述の実施例を参照）に鑑みれば、精子との共培養に供する細胞の由来が精子の由来と異なっていてもよい。精子との共培養に供する細胞の由来（生物種）の例として、ヒト、サル、ブタ、ウシ、ウマ、ヤギ、ヒツジ、イヌ、ネコ、マウス、ラット、モルモット、ハムスターを挙げることができる。また、精子の由来（生物種）の例は、ヒト、ブタ、ウシ、ウマ、ヤギ、ヒツジ、イヌ、ネコ、マウス、ラット、モルモット、ハムスター、鳥類（ニワトリ、ウズラ、カモなど）、魚類（サケ、マス、マグロ、カツオなど）である。一態様では、ヒト精子が用いられる。また、別の一態様では非ヒト哺乳動物精子（例えばブタ、ウシ、ウマ、ヤギ、ヒツジの精子）が用いられる。 The origin of the cell, that is, the biological species is not particularly limited, but the origin of the cell is determined in consideration of the origin of the sperm used in the method of the present invention and the use of the sperm (activated sperm) obtained by the method of the present invention. It is good to decide. For example, if the sperm is derived from a human and the activated sperm is used to treat human diseases, preferably human cells are subjected to co-culture with sperm. Thus, preferably, the co-cultured cell species and the sperm species are the same. However, in view of the surprising finding that the desired effect (ie, activation of sperm) by co-culture of specific cells and sperm is recognized across species (see Examples below for details), The origin of the cells subjected to the co-culture may be different from the origin of the sperm. Examples of the origin (species) of cells to be subjected to co-culture with sperm include humans, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, and hamsters. Examples of sperm origin (species) include humans, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, birds (chicken, quail, ducks, etc.), fish (salmons) , Trout, tuna, bonito, etc.). In one aspect, human sperm is used. In another embodiment, non-human mammal sperm (eg, pig, cow, horse, goat, sheep sperm) is used.
脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞の調製は常法に従えばよい。これらの細胞は各種用途に広く用いられており、当業者であれば文献や成書を参考にして容易に調製することができる。公的な細胞バンクから分譲された細胞や市販の細胞などを用いることにしてもよい。以下、細胞の調製法の例として、脂肪組織由来幹細胞の調製法と歯髄由来幹細胞の調製法を説明する。 Preparation of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells and fibroblasts may be performed according to a conventional method. These cells are widely used for various purposes, and those skilled in the art can easily prepare them with reference to literatures and books. Cells distributed from public cell banks or commercially available cells may be used. Hereinafter, as an example of a method for preparing cells, a method for preparing adipose tissue-derived stem cells and a method for preparing dental pulp-derived stem cells will be described.
＜脂肪組織由来幹細胞の調製法＞
本発明において「脂肪組織由来幹細胞（ASC）」とは、脂肪組織に含まれる体性幹細胞のことをいうが、多能性を維持している限りにおいて、当該体性幹細胞の培養（継代培養を含む）により得られる細胞も「脂肪組織由来幹細胞（ASC）」に該当するものとする。通常、ASCは、生体から分離された脂肪組織を出発材料とし、細胞集団（脂肪組織に由来する、ASC以外の細胞を含む）を構成する細胞として「単離された状態」に調製される。ここでの「単離された状態」とは、その本来の環境（即ち生体の一部を構成した状態）から取り出された状態、即ち人為的操作によって本来の存在状態と異なる状態で存在していることを意味する。尚、脂肪組織由来幹細胞はADRC（Adipose-derived regeneration cells）、AT-MSC（Adipose-derived mesenchymal stem cells）、AD-MSC（Adipose-derived mesenchymal stem cells）等とも呼ばれる。本明細書では以下の用語、即ち、脂肪組織由来幹細胞、ASC、ADRC、AT-MSC、AD-MSC、を相互に置換可能に使用する。
<Method for preparing adipose tissue-derived stem cells>
In the present invention, “adipose tissue-derived stem cells (ASC)” refers to somatic stem cells contained in adipose tissue. As long as pluripotency is maintained, the somatic stem cells are cultured (passaged). The cells obtained from the above are also “adipose tissue-derived stem cells (ASC)”. Usually, ASC is prepared in an “isolated state” as a cell constituting a cell population (including cells other than ASC derived from adipose tissue) using adipose tissue separated from a living body as a starting material. The “isolated state” as used herein means a state extracted from its original environment (that is, a state constituting a part of a living body), that is, a state different from the original existence state by an artificial operation. Means that Adipose tissue-derived stem cells are also referred to as ADRC (Adipose-derived regeneration cells), AT-MSC (Adipose-derived mesenchymal stem cells), AD-MSC (Adipose-derived mesenchymal stem cells), and the like. In the present specification, the following terms, that is, adipose tissue-derived stem cells, ASC, ADRC, AT-MSC, and AD-MSC are used interchangeably.
ASCは、脂肪基質からの幹細胞の分離、洗浄、濃縮、培養等の工程を経て調製される。ASCの調製法は特に限定されない。例えば公知の方法（Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr;24(4):150-4. Epub 2006 Feb 20. Review.; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr;7(2):211-28.等が参考になる）に従ってASCを調製することができる。また、脂肪組織からASCを調製するための装置（例えば、Celution（登録商標）装置（サイトリ・セラピューティクス社、米国、サンディエゴ））も市販されており、当該装置を利用してASCを調製することにしてもよい。当該装置を利用すると、脂肪組織より、ASCを含む細胞集団を分離できる（K. Lin. et al. Cytotherapy(2008) Vol. 10, No. 4, 417-426）。以下、ASCの調製法の具体例を示す。
ASC is prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix. A method for preparing ASC is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24 (4): 150-4. Epub 2006
（１）脂肪組織からの細胞集団の調製
脂肪組織は動物から切除、吸引などの手段で採取される。ここでの用語「動物」はヒト、及びヒト以外の哺乳動物（ペット動物、家畜、実験動物を含む。具体的には例えばサル、ブタ、ウシ、ウマ、ヤギ、ヒツジ、イヌ、ネコ、マウス、ラット、モルモット、ハムスター等）を含む。本発明の方法で得られた活性化精子を治療目的で使用する場合には、免疫拒絶の問題を回避するため、活性化精子を適用する対象（レシピエント）と同一の個体から脂肪組織（自己脂肪組織）を採取することが好ましい。但し、同種の動物の脂肪組織（他家）又は異種動物の脂肪組織の使用を妨げるものではない。
(1) Preparation of cell population from adipose tissue Adipose tissue is collected from animals by means such as excision and suction. The term “animal” herein includes humans and non-human mammals (pet animals, farm animals, laboratory animals. Specifically, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, Rat, guinea pig, hamster, etc.). When using the activated sperm obtained by the method of the present invention for therapeutic purposes, in order to avoid the problem of immune rejection, adipose tissue (self) from the same individual as the subject (recipient) to which the activated sperm is applied. It is preferable to collect (adipose tissue). However, this does not preclude the use of adipose tissue of the same species (other family) or adipose tissue of different species.
脂肪組織として皮下脂肪、内臓脂肪、筋肉内脂肪、筋肉間脂肪を例示できる。この中でも皮下脂肪は局所麻酔下で非常に簡単に採取できるため、採取の際のドナーへの負担が少なく、好ましい細胞源といえる。通常は一種類の脂肪組織を用いるが、二種類以上の脂肪組織を併用することも可能である。また、複数回に分けて採取した脂肪組織（同種の脂肪組織でなくてもよい）を混合し、以降の操作に使用してもよい。脂肪組織の採取量は、ドナーの種類や組織の種類、或いは必要とされるASCの量を考慮して定めることができ、例えば0.5〜500g程度である。ヒトをドナーとする場合にはドナーへの負担を考慮して一度に採取する量を約10〜20g以下にすることが好ましい。採取した脂肪組織は、必要に応じてそれに付着した血液成分の除去及び細片化を経た後、以下の酵素処理に供される。尚、脂肪組織を適当な緩衝液や培養液中で洗浄することによって血液成分を除去することができる。 Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, since subcutaneous fat can be collected very easily under local anesthesia, it can be said to be a preferable cell source with less burden on the donor during collection. Usually, one type of adipose tissue is used, but two or more types of adipose tissue can be used in combination. In addition, adipose tissue collected in multiple times (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ASC required, for example, about 0.5 to 500 g. When humans are used as donors, the amount collected at a time is preferably about 10 to 20 g or less in consideration of the burden on the donor. The collected adipose tissue is subjected to the following enzyme treatment after removal of blood components adhering to it and fragmentation as necessary. The blood component can be removed by washing the adipose tissue in an appropriate buffer or culture solution.
酵素処理は、脂肪組織をコラゲナーゼ、トリプシン、ディスパーゼ等の酵素によって消化することにより行う。このような酵素処理は当業者に既知の手法及び条件により実施すればよい（例えば、R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication参照）。以上の酵素処理によって得られた細胞集団は、多能性幹細胞、内皮細胞、間質細胞、血球系細胞、及び／又はこれらの前駆細胞等を含む。細胞集団を構成する細胞の種類や比率などは、使用した脂肪組織の由来や種類に依存する。 Enzymatic treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase and the like. Such enzyme treatment may be performed by techniques and conditions known to those skilled in the art (see, for example, RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . The cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, stromal cells, blood cells, and / or precursor cells thereof. The type and ratio of the cells constituting the cell population depend on the origin and type of the adipose tissue used.
（２）沈降細胞集団（SVF画分：stromal vascular fractions）の取得
細胞集団は続いて遠心処理に供される。遠心処理による沈渣を沈降細胞集団（本明細書では「SVF画分」ともいう）として回収する。遠心処理の条件は、細胞の種類や量によって異なるが、例えば1〜10分間、800〜1500rpmである。尚、遠心処理に先立ち、酵素処理後の細胞集団をろ過等に供し、その中に含まれる酵素未消化組織等を除去しておくことが好ましい。
(2) Acquisition of sedimented cell population (SVF fraction: stroma vascular fractions) The cell population is subsequently subjected to centrifugation. The sediment by centrifugation is collected as a sedimented cell population (also referred to herein as “SVF fraction”). The conditions for the centrifugation process vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm. Prior to centrifugation, the cell population after the enzyme treatment is preferably subjected to filtration or the like, and the enzyme undigested tissue contained therein is preferably removed.
ここで得られた「SVF画分」はASCを含む。従って、SVF画分を、精子との共培養に使用する細胞として用いることもできる。尚、SVF画分を構成する細胞の種類や比率などは、使用した脂肪組織の由来や種類、酵素処理の条件などに依存する。また、国際公開第２００６／００６６９２Ａ１号パンフレットにはSVF画分の特徴が示されている。 The “SVF fraction” obtained here contains ASC. Therefore, the SVF fraction can also be used as cells used for co-culture with sperm. The type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions for enzyme treatment, and the like. In addition, the characteristics of the SVF fraction are shown in the pamphlet of International Publication No. 2006 / 006692A1.
（３）接着性細胞（ASC）の選択培養及び細胞の回収
SVF画分にはASCの他、他の細胞成分（内皮細胞、間質細胞、血球系細胞、これらの前駆細胞等）が含まれる。そこで本発明の一態様では以下の選択培養を行い、SVF画分から不要な細胞成分を除去する。そして、その結果得られた細胞をASCとして本発明に用いる。
(3) Selective culture of adherent cells (ASC) and cell recovery
In addition to ASC, the SVF fraction contains other cell components (endothelial cells, stromal cells, blood cells, progenitor cells thereof, etc.). Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The resulting cells are used as ASC in the present invention.
まず、SVF画分を適当な培地に懸濁した後、培養皿に播種し、一晩培養する。培地交換によって浮遊細胞（非接着性細胞）を除去する。その後、適宜培地交換（例えば２〜４日に一度）をしながら培養を継続する。必要に応じて継代培養を行う。継代数は特に限定されないが、多能性と増殖能力の維持の観点からは過度に継代を繰り返すことは好ましくない（５継代程度までに留めておくことが好ましい）。尚、培養用の培地には、通常の動物細胞培養用の培地を使用することができる。例えば、Dulbecco's modified Eagle's Medium(DMEM)（日水製薬株式会社等）、α-MEM(大日本製薬株式会社等)、DMEM:Ham's F12混合培地(1:1)(大日本製薬株式会社等)、Ham's F12 medium(大日本製薬株式会社等)、MCDB201培地(機能性ペプチド研究所)等を使用することができる。血清（ウシ胎仔血清、ヒト血清、羊血清など）又は血清代替物（Knockout serum replacement(KSR)など）を添加した培地を使用することにしてもよい。血清又は血清代替物の添加量は例えば5%(v/v)〜30%(v/v)の範囲内で設定可能である。 First, the SVF fraction is suspended in an appropriate medium, seeded on a culture dish, and cultured overnight. Suspension cells (non-adherent cells) are removed by medium exchange. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 2 to 4 days). Subculture as necessary. The number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat the passages excessively (preferably to be kept to about 5 passages). As the culture medium, a normal animal cell culture medium can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used. A medium supplemented with serum (fetal calf serum, human serum, sheep serum, etc.) or a serum substitute (Knockout serum replacement (KSR), etc.) may be used. The addition amount of serum or serum replacement can be set, for example, within a range of 5% (v / v) to 30% (v / v).
以上の操作によって接着性細胞が選択的に生存・増殖する。続いて、増殖した細胞を回収する。回収操作は常法に従えばよく、例えば酵素処理（トリプシンやディスパーゼ処理）後の細胞をセルスクレイパーやピペットなどで剥離することによって容易に回収することができる。また、市販の温度感受性培養皿などを用いてシート培養した場合は、酵素処理をせずにそのままシート状に細胞を回収することも可能である。このようにして回収した細胞（ASC）を用いることにより、ASCを高純度で含有する細胞集団を調製することができる。 By the above operation, adherent cells selectively survive and proliferate. Subsequently, the proliferated cells are collected. The collection operation may be carried out in accordance with a conventional method. For example, the cells after enzyme treatment (trypsin or dispase treatment) can be easily collected by detaching them with a cell scraper or pipette. In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish or the like, it is also possible to recover the cells as they are without performing enzyme treatment. By using the cells (ASC) collected in this manner, a cell population containing ASC with high purity can be prepared.
（４）低血清培養（低血清培地での選択的培養）及び細胞の回収
本発明の一態様では、上記（３）の操作の代わりに又は上記（３）の操作の後に以下の低血清培養を行う。そして、その結果得られた細胞をASCとして本発明に用いる。
(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one embodiment of the present invention, the following low-serum culture is performed instead of or after the operation of (3) above. I do. The resulting cells are used as ASC in the present invention.
低血清培養では、SVF画分（（３）の後にこの工程を実施する場合には（３）で回収した細胞を用いる）を低血清条件下で培養し、目的の多能性幹細胞（即ちASC）を選択的に増殖させる。低血清培養法では用いる血清が少量で済むことから、本発明の方法で得られた活性化精子を治療目的に使用する場合、対象（患者）自身の血清を使用することが可能となる。即ち、自己血清を用いた培養が可能となる。ここでの「低血清条件下」とは5%以下の血清を培地中に含む条件である。好ましくは2%(V/V)以下の血清を含む培養液中で細胞培養する。更に好ましくは、2%(V/V)以下の血清と1〜100ng/mlの線維芽細胞増殖因子-2（bFGF）を含有する培養液中で細胞培養する。 In low serum culture, the SVF fraction (when using this step after (3), the cells collected in (3) are used) are cultured under low serum conditions, and the target pluripotent stem cell (ie ASC ) Selectively. Since a small amount of serum is used in the low serum culture method, when the activated sperm obtained by the method of the present invention is used for therapeutic purposes, the subject's (patient) 's own serum can be used. That is, culture using autoserum becomes possible. Here, “under low serum conditions” is a condition containing 5% or less of serum in the medium. The cells are preferably cultured in a culture solution containing 2% (V / V) or less of serum. More preferably, the cells are cultured in a culture solution containing 2% (V / V) or less of serum and 1 to 100 ng / ml of fibroblast growth factor-2 (bFGF).
血清はウシ胎仔血清に限られるものではなく、ヒト血清や羊血清等を用いることができる。本発明の方法で得られた活性化精子をヒトの治療に使用する場合には、好ましくはヒト血清、更に好ましくは治療対象の血清（即ち自己血清）を用いる。 Serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can be used. When the activated sperm obtained by the method of the present invention is used for human therapy, preferably human serum, more preferably serum to be treated (ie, autoserum) is used.
培地は、使用の際に含有する血清量が低いことを条件として、通常の動物細胞培養用の培地を使用することができる。例えば、Dulbecco's modified Eagle's Medium(DMEM)（日水製薬株式会社等）、α-MEM(大日本製薬株式会社等)、DMEM:Ham's F12混合培地(1:1)(大日本製薬株式会社等)、Ham's F12 medium(大日本製薬株式会社等)、MCDB201培地(機能性ペプチド研究所)等を使用することができる。 As a medium, a normal medium for animal cell culture can be used on the condition that the amount of serum contained in use is low. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd.), α-MEM (Dainippon Pharmaceutical Co., Ltd.), DMEM: Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Research Institute), etc. can be used.
以上の方法で培養することによって、多能性幹細胞（ASC）を選択的に増殖させることができる。また、上記の培養条件で増殖する多能性幹細胞（ASC）は高い増殖活性を持つので、継代培養によって、本発明に必要とされる数の細胞を容易に調製することができる。尚、国際公開第２００６／００６６９２Ａ１号パンフレットには、SVF画分を低血清培養することによって選択的に増殖する細胞の特徴が示されている。 By culturing by the above method, pluripotent stem cells (ASC) can be selectively proliferated. In addition, since pluripotent stem cells (ASC) that proliferate under the above culture conditions have high proliferation activity, the number of cells required for the present invention can be easily prepared by subculture. In addition, International Publication No. 2006 / 006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction in low serum.
続いて、上記の低血清培養によって選択的に増殖した細胞を回収する。回収操作は上記（３）の場合と同様に行えばよい。回収した細胞（ASC）を用いることにより、ASCを高純度で含有する細胞集団を得ることができる。 Subsequently, the cells selectively proliferated by the low serum culture are collected. The collection operation may be performed in the same manner as in the above (3). By using the collected cells (ASC), a cell population containing ASC with high purity can be obtained.
以上の方法では、SVF画分を低血清培養して増殖した細胞が利用に供されることになるが、脂肪組織から得た細胞集団を直接（SVF画分を得るための遠心処理を介することなく）低血清培養することによって増殖した細胞をASCとして用いることにしてもよい。即ち本発明の一態様では、脂肪組織から得た細胞集団を低血清培養したときに増殖した細胞をASCとして用いる。また、選択的培養（上記（３）及び（４））によって得られる多能性幹細胞ではなく、SVF画分（脂肪組織由来間葉系幹細胞を含有する）をそのまま用いることにしてもよい。尚、ここでの「そのまま用いて」とは、選択的培養を経ることなく本発明に用いること、を意味する。 In the above method, cells proliferated by low serum culture of the SVF fraction will be used, but the cell population obtained from the adipose tissue is directly used (through centrifugation to obtain the SVF fraction). (Not) Cells grown by low serum culture may be used as ASC. That is, in one embodiment of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low serum are used as ASC. Further, instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)), the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as it is. Here, “use as it is” means to use in the present invention without undergoing selective culture.
＜歯髄由来幹細胞の調製方法＞
幹細胞のソースとして歯髄が注目されており、自己増殖能と多分化能を併せ持つ新規な幹細胞集団として、永久歯の歯髄由来幹細胞（dental pulp stem cells: DPSCs）と乳歯の歯髄由来幹細胞（stem cells from exfoliated deciduous teeth; SHED）が同定されている（S. Gronthos, M. Mankani, J. Brahim, P. G. Robey, S. Shi, Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13625-30.；M. Miura, S. Gronthos, M. Zhao, B. Lu, L. W. Fisher, P. G. Robey, S. Shi, SHED: Stem cells from human exfoliated deciduous teeth, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 5807-12.；国際公開第２００６／０１０６００号パンフレット）。
<Preparation method of dental pulp-derived stem cells>
The dental pulp is attracting attention as a source of stem cells, and as a new stem cell population that has both self-proliferating ability and multipotency, dental pulp stem cells (DPSCs) of permanent teeth and stem cells from exfoliated of deciduous teeth deciduous teeth (SHED) have been identified (S. Gronthos, M. Mankani, J. Brahim, PG Robey, S. Shi, Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo, Proc. Natl. Acad Sci. USA 97 (2000) 13625-30 .; M. Miura, S. Gronthos, M. Zhao, B. Lu, LW Fisher, PG Robey, S. Shi, SHED: Stem cells from human exfoliated deciduous teeth, Proc Natl. Acad. Sci. USA 100 (2003) 5807-12 .; WO 2006/010600 pamphlet).
歯髄幹細胞の採取・調製法の一例を以下に示す。この採取・調製法では（１）歯髄の採取、（２）酵素処理、（３）細胞培養、（４）細胞の回収を順に行う。 An example of a method for collecting and preparing dental pulp stem cells is shown below. In this collection / preparation method, (1) collection of dental pulp, (2) enzyme treatment, (3) cell culture, and (4) collection of cells are sequentially performed.
（１）歯髄の採取
自然に脱落した乳歯（又は抜歯した乳歯、或いは永久歯）をクロロヘキシジンまたはイソジン溶液で消毒した後、歯冠部を分割し歯科用リーマーにて歯髄組織を回収する。
(1) Collection of dental pulp After naturally deciduous deciduous teeth (or extracted deciduous teeth or permanent teeth) are sterilized with a chlorohexidine or isodine solution, the crown portion is divided and the pulp tissue is collected with a dental reamer.
（２）酵素処理
採取した歯髄組織を基本培地（10%ウシ血清・抗生物質含有ダルベッコ変法イーグル培地）に懸濁し、2mg/mlのコラゲナーゼ及びディスパーゼで37℃、1時間処理する。5分間の遠心操作（5000回転／分）により酵素処理後の歯髄細胞を回収する。セルストレーナーによる細胞選別はSHEDやDPSCの神経幹細胞分画の回収効率を低下させるので原則、使用しない。
(2) Enzyme treatment The collected dental pulp tissue is suspended in a basic medium (10% bovine serum / antibiotic Dulbecco's modified Eagle medium) and treated with 2 mg / ml collagenase and dispase at 37 ° C. for 1 hour. The pulp cells after enzyme treatment are collected by centrifugation for 5 minutes (5000 rpm). In principle, cell sorting with a cell strainer is not used because it reduces the collection efficiency of neural stem cell fractions of SHED and DPSC.
（３）細胞培養
細胞を4cc基本培地で再懸濁し、直径6cmの付着性細胞培養用ディッシュに播種する。5％CO2、37℃に調整したインキュベータにて3日間培養した後、コロニーを形成した接着性細胞を0.05%トリプシン・EDTAにて5分間、37℃で処理する。ディッシュから剥離した歯髄細胞を直径10cmの付着性細胞培養用ディッシュに播種し拡大培養を行う。例えば、肉眼で観察してサブコンフルエント（培養容器の表面の約70％を細胞が占める状態）又はコンフルエントに達したときに細胞を培養容器から剥離して回収し、再度、培養液を満たした培養容器に播種する。継代培養を繰り返し行ってもよい。例えば継代培養を１〜８回行い、必要な細胞数（例えば約1×107個/ml）まで増殖させる。尚、培養容器からの細胞の剥離は、トリプシン処理など常法で実施することができる。以上の培養の後、細胞を回収して保存することにしてもよい（保存条件は例えば-198℃）。
(3) Cell culture Cells are resuspended in 4 cc basal medium and seeded in 6 cm diameter adherent cell culture dishes. After culturing in an incubator adjusted to 5% CO 2 and 37 ° C. for 3 days, the adherent cells that formed colonies are treated with 0.05% trypsin · EDTA for 5 minutes at 37 ° C. The dental pulp cells detached from the dish are seeded in an adherent cell culture dish having a diameter of 10 cm and expanded. For example, when observing with the naked eye, it reaches the sub-confluent state (a state in which the cells occupy about 70% of the surface of the culture container) or confluent, and the cells are detached from the culture container and collected, and the culture is filled again with the culture solution. Seed in containers. Subculturing may be repeated. For example, the subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × 10 7 cells / ml). The cell can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and stored (storage conditions are, for example, -198 ° C.).
（４）細胞の回収
次に、細胞を回収する。トリプシン処理等で培養容器から細胞を剥離した後、遠心処理を施すことによって細胞（歯髄由来幹細胞）を回収することができる。
(4) Cell recovery Next, cells are recovered. Cells (dental pulp-derived stem cells) can be recovered by centrifuging after detaching the cells from the culture vessel by trypsin treatment or the like.
共培養用細胞（脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞）と精子を共培養するため、本発明の一態様では、以上のようにして調製ないし用意した共培養用細胞と精子を培養容器に播種し、培養に供する。播種する順序は特に問わず、いずれを先に播種してもよい。また、両者を混合した後に播種することにしてもよい。培養条件は、動物細胞の培養において一般に採用されている条件とすればよい。即ち、例えば３６℃〜３９℃、５%CO2の環境下で培養すればよい。但し、精子の動物種を考慮し、動物の体温に近い温度に設定することが好ましい。例えばブタやウシなどの家畜の精子を用いる場合、比較的高温（３８℃〜３９℃）に設定するとよい。また、ヒトの精子であれば３７℃前後の温度条件が好ましい。基本培地としてダルベッコ変法イーグル培地（D-MEM）（ナカライテスク株式会社、シグマ社、Gibco社等）、ハムF12培地（HamF12）（SIGMA社、Gibco社等）、イスコフ改変ダルベッコ培地（IMDM）（GIBCO社等）、グラスゴー基本培地（Gibco社等）、RPMI1640培地等を用いることができる。二種以上の基本培地を併用することにしてもよい。培地に添加可能な成分の例として血清、血漿、血清アルブミン、ポリビニルアルコール（PVA）、ポリビニルピロリドン（PVP）、抗生物質、2-メルカプトエタノール、アミノ酸、ビタミン、無機塩を挙げることができる。典型的には培養皿などの培養容器を用いて二次元的に細胞を培養する。共培養の期間は例えば１時間〜１日、好ましくは２時間〜５時間とする。当該培養期間が短すぎると、期待される効果（精子の活性化）が十分に得られない。他方、当該培養期間が長すぎると、精子の活性低下を引き起こし得る。 In order to co-cultivate cells for co-culture (adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells or fibroblasts) and sperm, in one embodiment of the present invention, the co-culture cells prepared or prepared as described above And sperm are seeded in a culture vessel and used for culture. The order of seeding is not particularly limited, and either may be seeded first. Moreover, you may decide to sow after mixing both. The culture conditions may be those generally employed for animal cell culture. That is, for example, it may be cultured in an environment of 36 ° C. to 39 ° C. and 5% CO 2 . However, considering the species of sperm, it is preferable to set the temperature close to the body temperature of the animal. For example, when using sperm of livestock such as pigs and cows, it is preferable to set the temperature to a relatively high temperature (38 ° C. to 39 ° C.). For human sperm, a temperature condition of around 37 ° C. is preferable. Dulbecco's modified Eagle medium (D-MEM) (Nacalai Tesque, Sigma, Gibco, etc.), Ham F12 medium (SamMA, Gibco, etc.), Iscov modified Dulbecco medium (IMDM) (basic medium) GIBCO, etc.), Glasgow basic medium (Gibco, etc.), RPMI1640 medium, etc. can be used. Two or more basic media may be used in combination. Examples of components that can be added to the medium include serum, plasma, serum albumin, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), antibiotics, 2-mercaptoethanol, amino acids, vitamins, and inorganic salts. Typically, cells are cultured two-dimensionally using a culture vessel such as a culture dish. The period of co-culture is, for example, 1 hour to 1 day, preferably 2 hours to 5 hours. If the culture period is too short, the expected effect (activation of sperm) cannot be obtained sufficiently. On the other hand, if the culture period is too long, it may cause a decrease in sperm activity.
本発明の別の一態様では、層状（シート状）に形成した共培養用細胞の上で精子を培養する。この態様の場合、共培養用細胞と精子の共培養に先立って、層状の共培養細胞を用意しておく。例えば、共培養用細胞を適当な培養容器（典型的には培養皿）で培養し、増殖させることにより共培養用細胞層（共培養用細胞シート）を得た後、当該細胞層上に精子を播種して培養する。このように、共培養用細胞層の形成と精子の共培養を連続的に実施するのではなく、共培養用細胞層を事前に用意しておき（例えば、細胞層形成後に保存しておく）、精子との共培養に利用することにしてもよい。このような実施形態の場合、市販の又は公的な細胞バンクなどから分譲を受けた細胞層を利用してもよい。 In another embodiment of the present invention, sperm is cultured on cells for co-culture formed in a layered form (sheet form). In this embodiment, layered co-culture cells are prepared prior to co-culture of co-culture cells and sperm. For example, after coculturing cells are cultured in an appropriate culture container (typically a culture dish) and proliferated to obtain a cell layer for coculture (cell sheet for coculture), sperm is then deposited on the cell layer. Is seeded and cultured. Thus, instead of continuously forming the cell layer for co-culture and co-culturing sperm, the cell layer for co-culture is prepared in advance (for example, stored after the cell layer is formed). It may be used for co-culture with sperm. In such an embodiment, a cell layer obtained from a commercially available or public cell bank may be used.
前述のように、本発明者らの検討によって、細胞と精子の接触によって精子が活性化することが判明した。従って、本発明における共培養では、共培養用細胞と精子が接触する状態を形成することが重要である。層状の共培養用細胞を用いる上記態様は、共培養用細胞と精子が接触する環境を作り出すことに有利である。 As described above, the inventors' study has revealed that sperm is activated by contact between cells and sperm. Therefore, in the co-culture in the present invention, it is important to form a state in which cells for co-culture and sperm are in contact. The above-described embodiment using the layered co-culture cells is advantageous for creating an environment where the co-culture cells and sperm are in contact.
共培養用細胞との共培養によって精子が活性化される。即ち、活性化された精子が得られる。本発明の一態様では、共培養後の精子（即ち、活性化された精子）を回収する（ステップ（２））。このとき、好ましくは、共培養用細胞から分離した状態で精子を回収する。層状の共培養用細胞を用いると、このような分離回収を比較的容易に行うことができる。回収した精子は後述の各種用途に用いられる。 Sperm are activated by co-culture with cells for co-culture. That is, activated sperm is obtained. In one embodiment of the present invention, sperm after co-culture (that is, activated sperm) is collected (step (2)). At this time, preferably, spermatozoa are collected in a state separated from the cells for co-culture. Such separation and recovery can be performed relatively easily by using layered co-culture cells. The collected sperm is used for various uses described later.
２．活性化精子の用途
本発明の第２の局面は、本発明の方法で得られた活性化精子の用途に関する。活性化精子は、精子障害を伴う又は精子障害が主因又は副因の各種疾患（例えば、精索静脈瘤、男子不妊症、停留精巣、X線照射、悪性腫瘍術後又は化学療法後の精子障害）の治療・改善、人工授精（成功率や効率の向上）、家畜の繁殖（人工授精の成功率や効率の向上）、育種・種の維持（例えば絶滅危惧種の維持、ペットの系統の維持又は交雑）等に利用され得る。
2. Use of activated sperm The second aspect of the present invention relates to the use of activated sperm obtained by the method of the present invention. Activated sperm is a variety of diseases associated with or associated with sperm disorders (eg varicocele, male infertility, cryptorchidism, X-ray irradiation, post-malignant surgery or post-chemotherapy sperm disorder ) Treatment / improvement, artificial insemination (improving success rate and efficiency), livestock breeding (improving success rate and efficiency of artificial insemination), breeding / species maintenance (eg maintaining endangered species, maintaining pet lines) Or cross).
本発明の活性化精子を用いて人工授精を実施する場合（家畜の繁殖や育種・種の維持を目的とした人工授精も含む）、生体内又は生体外で活性化精子と卵子（卵細胞、卵）が共存する状態を形成させることになる。生体内で共存状態を形成させるためには、活性化精子を卵子が存在する生体の部位に投与する。例えば、哺乳動物であれば子宮内に活性化精子を注入する。他方、生体外で共存状態を形成させるのであれば、生体からの単離や細胞バンクなどから分譲などによって用意した卵子と活性化精子を同一の容器内に入れ、インキュベートすればよい。本発明に特徴的な条件、即ち、本発明の活性化方法で得た活性化精子を使用すること以外については常法に従えばよい（例えば、牛の人工授精マニュアル（社団法人 畜産技術協会）、馬人工授精マニュアル（一般社団法人 日本家畜人工授精師協会）、牛の繁殖技術マニュアル（一般社団法人 日本家畜人工授精師協会）、家畜人工授精ハンドブック（一般社団法人 日本家畜人工授精師協会）等が参考になる）。 When artificial insemination is performed using the activated sperm of the present invention (including artificial insemination for the purpose of breeding and breeding / maintenance of livestock), activated sperm and eggs (egg cells, eggs) in vivo or in vitro ) Will coexist. In order to form a coexistence state in a living body, activated sperm is administered to a part of a living body where an egg is present. For example, in the case of a mammal, activated sperm is injected into the uterus. On the other hand, if a coexistence state is formed outside the living body, an egg and activated sperm prepared by isolation from a living body or by distribution from a cell bank or the like may be placed in the same container and incubated. Except for using the conditions characteristic of the present invention, that is, using the activated spermatozoa obtained by the activation method of the present invention, a normal method may be followed (for example, an artificial insemination manual for cattle (Japan Society for Animal Husbandry) , Horse artificial insemination manual (Japan Livestock Artificial Insemination Association), cattle breeding technical manual (Japan Livestock Artificial Insemination Association), livestock artificial insemination handbook (Japan Livestock Artificial Insemination Association), etc. Is helpful).
本発明は、人工授精方法の一態様として、本発明の方法（精子の活性化方法）を適用していない精子（典型的には、生体から分離した精子）を用いた人工授精方法も提供する。この態様では、脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞（説明の便宜上、本発明の第１の局面における呼称に統一して「共培養用細胞」と呼ぶ）、精子、及び卵子の三者が共存する状態を形成し、精子の活性化と受精を進行させる。「共存」の具体的方法の例は、（１）共培養用細胞とともに精子を、卵子が存在する生体の部位に投与する方法（例えば非ヒト哺乳動物の子宮に注入する）、（２）生体外において、共培養用細胞と卵子を混合し、そこへ精子を添加する方法、（３）生体外において、共培養用細胞と精子を混合し、そこへ卵子を添加する方法、（４）生体外において、共培養用細胞、精子、卵子を混合する方法、（５）生体外において、共培養用細胞と精子を共培養（即ち本発明の精子活性化方法を実施）した後、卵子を添加する方法、である。尚、（２）〜（５）は体外受精方法に該当する。 As one aspect of the artificial insemination method, the present invention also provides an artificial insemination method using sperm (typically, sperm separated from a living body) to which the method of the present invention (sperm activation method) is not applied. . In this embodiment, adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells, or fibroblasts (for convenience of explanation, the names in the first aspect of the present invention are referred to as “co-culture cells”), sperm, And the three of the eggs coexist, and the activation and fertilization of sperm are advanced. Examples of specific methods of “coexistence” are (1) a method of administering sperm together with cells for co-culture to a living body site where an egg is present (for example, injecting into the uterus of a non-human mammal), (2) living body A method of mixing coculture cells and ova outside and adding sperm thereto; (3) a method of mixing coculture cells and sperm outside the organism and adding eggs thereto; and (4) living body. A method of mixing cells for culturing, sperm, and ovum outside, (5) Co-culture of cells for culturing with sperm in vitro (that is, carrying out the sperm activation method of the present invention), and then adding an egg How to do. Incidentally, (2) to (5) correspond to in vitro fertilization methods.
本発明は様々な生物種を対象とする。精子又は卵子の由来となる生物種の例はヒト、ブタ、ウシ、ウマ、ヤギ、ヒツジ、鳥類（ニワトリ、ウズラ、カモなど）、魚類（サケ、マス、マグロ、カツオなど）である。精子と卵子の生物種は原則として同一である。但し、受精可能な組合せであれば、精子の生物種と卵子の生物種が異なっていてもよい。 The present invention is directed to various biological species. Examples of biological species from which sperm or eggs are derived are humans, pigs, cows, horses, goats, sheep, birds (chicken, quail, ducks, etc.), and fish (salmon, trout, tuna, skipjack etc.). The species of sperm and egg are in principle the same. However, as long as it is a fertilizable combination, the sperm species and the ovum species may be different.
上記の各用途への利用に際し、活性化精子を組成物の形態で提供することもできる。即ち、本発明は、活性化精子を含有する組成物（本明細書において「活性化精子組成物」と呼ぶ）も提供する。細胞の保護を目的としてジメチルスルフォキシド（DMSO）や血清アルブミン等を、細菌の混入を阻止することを目的として抗生物質等を、細胞の活性化、増殖又は分化誘導などを目的として各種の成分（ビタミン類、サイトカイン、成長因子、ステロイド等）を本発明の精子活性化剤に含有させてもよい。さらに、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を本発明の活性化精子組成物に含有させてもよい。 For use in each of the above applications, activated sperm can be provided in the form of a composition. That is, the present invention also provides a composition containing activated sperm (referred to herein as an “activated sperm composition”). Dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of cell protection, antibiotics, etc. for the purpose of preventing bacterial contamination, and various components for the purpose of cell activation, proliferation or differentiation induction, etc. (Vitamins, cytokines, growth factors, steroids, etc.) may be included in the sperm activator of the present invention. In addition, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) You may make it contain in the activated sperm composition of this invention.
ここで、脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞（即ち共培養用細胞）との接触が精子の活性化に有効であるとの知見に従えば、期待される生理作用が発揮される前に、共培養用細胞との接触状態を形成しておくことが有利且つ重要であるといえる。そこで、本発明の一態様では、本発明の方法（精子の活性化方法）を適用していない精子（典型的には、生体から分離した精子）と共培養用細胞を混合し、活性化精子組成物を得る。従って、この態様の組成物には、精子と共培養用細胞が含有されることになる。 Here, according to the knowledge that contact with adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells or fibroblasts (ie, cells for co-culture) is effective for sperm activation, expected physiological effects It can be said that it is advantageous and important to form a contact state with the cell for co-culture before the effect is exhibited. Thus, in one embodiment of the present invention, sperm (typically, sperm separated from a living body) to which the method of the present invention (method for activating sperm) is not applied and a cell for co-culture are mixed, and activated sperm A composition is obtained. Therefore, the composition of this embodiment contains sperm and cells for co-culture.
３．精子活性化剤
前述の通り、本発明者らの検討によって、脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞との接触状態を形成することによって精子が活性化することが明らかとなった。当該知見に基づき、本発明は更に、脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞を有効成分として含有する精子活性化剤を提供する。本発明の精子活性化剤は、上記の活性化精子組成物と同様に、精子障害を伴う又は精子障害が主因又は副因の各種疾患の治療・改善、人工受精、家畜の繁殖、育種・種の維持等に利用され得る。但し、精子障害を伴う又は精子障害が主因又は副因の各種疾患の治療・改善に対して特に有効である。当該用途に使用する場合には、治療上有効量の細胞が投与されるように、一回投与分の量として例えば1x104個〜1x107個の細胞を含有させるとよい。細胞の含有量は、対象疾患、適用対象（レシピエント）の性別、年齢、体重、患部の状態、細胞の状態などを考慮して適宜調整することができる。また、投与スケジュールは、対象（患者）の性別、年齢、体重、病態などを考慮して作成すればよい。単回投与の他、連続的又は定期的に複数回投与することにしてもよい。複数回投与する際の投与間隔は特に限定されず、例えば１日〜１月である。また、投与回数も特に限定されない。投与回数の例は２回〜１０回である。
3. Sperm Activator As described above, the inventors' studies have revealed that sperm is activated by forming a contact state with adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells or fibroblasts. became. Based on this finding, the present invention further provides a sperm activator containing adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells or fibroblasts as an active ingredient. The sperm activator of the present invention is similar to the above-mentioned activated sperm composition, treatment / amelioration of various diseases accompanied by sperm disorder or mainly caused by or associated with sperm disorder, artificial fertilization, breeding of livestock, breeding / species It can be used for maintenance of However, it is particularly effective for the treatment and improvement of various diseases accompanied by sperm disorder or caused mainly by or associated with sperm disorder. When used in this application, for example, 1 × 10 4 to 1 × 10 7 cells may be contained as a single dose so that a therapeutically effective amount of cells is administered. The cell content can be appropriately adjusted in consideration of the target disease, the sex of the application target (recipient), age, weight, state of the affected area, cell state, and the like. Moreover, what is necessary is just to create an administration schedule in consideration of sex, age, weight, disease state, etc. of the subject (patient). In addition to single administration, multiple administration may be performed continuously or periodically. The administration interval when administering multiple times is not particularly limited, and is, for example, 1 day to 1 month. Moreover, the frequency | count of administration is not specifically limited. Examples of the number of administrations are 2 to 10 times.
活性化精子組成物の場合と同様に、各種任意成分（細胞の保護剤、抗生物質、ビタミン、サイトカイン、成長因子、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水など）を含有させてもよい。尚、特に言及しない点（各細胞の動物種、精子の動物種など）については、本発明の第２の局面の場合と同様であり、対応する説明を援用する。 As in the case of the activated sperm composition, various optional components (cell protective agents, antibiotics, vitamins, cytokines, growth factors, carriers, excipients, disintegrants, buffers, emulsifiers, suspension agents, soothing agents Agents, stabilizers, preservatives, preservatives, physiological saline, etc.). Note that points not specifically mentioned (animal species of each cell, sperm animal species, etc.) are the same as in the case of the second aspect of the present invention, and corresponding explanations are cited.
本発明の精子活性化剤は、その有効成分（脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞又は線維芽細胞）と精子が接触する状態を生体内又は生体外で形成させるために使用される。当該接触状態を生体内で形成させるためには、本発明の精子活性化剤を生体に投与することになる。典型的には、精子の貯留器官である精嚢に注入することになるが、投与後に本発明の精子活性化剤の有効成分と精子が接触する状態が形成される限りにおいて、投与部位や投与方法は特に限定されない。一方、本発明の精子活性化剤の有効成分と精子との接触状態を生体外で形成させるためには、例えば、予め用意しておいた精子と本発明の精子活性化剤を適当な容器（典型的には培養皿、培養フラスコなど）に入れればよい。添加順序は特に問わず、いずれを先に入れることにしてもよい。また、両者を混合した後に容器に移すことにしてもよい。 The sperm activator of the present invention is used to form a state where its active ingredient (adipose tissue-derived stem cell, bone marrow-derived stem cell, dental pulp-derived stem cell or fibroblast) is in contact with sperm in vivo or in vitro. . In order to form the contact state in vivo, the sperm activator of the present invention is administered to the living body. Typically, it will be injected into the seminal vesicle, which is a sperm storage organ, but as long as the sperm comes into contact with the active ingredient of the sperm activator of the present invention after administration, the site of administration and administration The method is not particularly limited. On the other hand, in order to form the contact state between the active ingredient of the sperm activator of the present invention and the sperm in vitro, for example, the sperm prepared in advance and the sperm activator of the present invention are combined in an appropriate container ( Typically, it may be placed in a culture dish, a culture flask or the like. The order of addition is not particularly limited, and either may be added first. Moreover, you may decide to move to a container, after mixing both.
１．脂肪組織由来幹細胞（ASC）の精子活性化効果
（１）目的及び方法
臨床では陰嚢静脈逆流または鬱滞により精巣温度が上昇し、精子運動率が低下する精索静脈瘤の病態がある。また、獣医学では夏の室内温度上昇のために上記同様な病態で、いわゆる夏季不妊症と呼ばれるブタ精子の運動率低下が毎年夏季に発生し、人工授精用精液の供給に不安定性を来している。ヒト精索静脈瘤において精子運動性の低下により発生する男子不妊症を治療するための新規細胞療法を開発する目的で、本疾患と同一の病態を持つブタ夏季不妊症の精子とブタ脂肪幹細胞（ASC）を利用して共培養による治療を試みた。さらに、ブタ精子に及ぼすヒト幹細胞の治療効果を調べた。尚、準備実験として、牛精子とブタASCとの共培養を行った。以下、実験プロトコールを示す。
(i) ブタASCをシート状あるいは浮遊した状態でブタ精子と培養した（ASCと精子の共培養）。
(ii) 培養液として0.1% HSAを添加したPBS(-)あるいはDMEM/Ham’s F12培養液（図中では「培地」と表記）を使用し、コントロール群ではこれらの中にブタ精子のみを浮遊して培養した。共培養群では、シート状（図中では「ASC」と表記）又は浮遊した状態のブタ脂肪幹細胞（図中では「ASC.sus」と表記）とブタ精子を共培養した。
(iii) 培養開始後、0時間（コントロール群のみ）及び5時間後に精子を取り出し、血球計算盤（C-Chip）に入れ、その運動性を倒立位相差顕微鏡で観察すると共に、精子の運動を20秒間撮影した。
(iv) 撮影した精子の運動を解析し、数えた総精子数に対する運動している精子の割合（運動率，％ Motile spermatozoa）および運動している精子の数に対する前進運動する精子の数の比率（前進運動率，％ Progressively motile spermatozoa）を算出した。
(v) 2回繰り返した結果を平均値±標準誤差で示した。処置および培養時間が運動率と前進運動率へ及ぼす影響を2元配置分散分析法で解析した後、交互作用に有意差が認められたので、各時間における処置間の有意差をテューキーの多重比較検定（Tukey’s multiple comparison test）により検定した。
1. Sperm activation effect of adipose tissue-derived stem cells (ASC) (1) Purpose and method Clinically, there is a pathological condition of varicocele of the spermatic cord that increases testicular temperature and decreases sperm motility due to scrotal vein reflux or stagnation. In addition, in veterinary medicine, due to a rise in indoor room temperature in summer, a decrease in the motility rate of porcine sperm, so-called summer infertility, occurs in summer every year, causing instability in the supply of semen for artificial insemination. ing. In order to develop a new cell therapy to treat male infertility caused by decreased sperm motility in human varicocele, sperm and porcine adipose stem cells of pig summer infertility with the same pathology as this disease ( ASC was used to try co-culture treatment. Furthermore, the therapeutic effect of human stem cells on porcine sperm was investigated. As a preparatory experiment, co-culture of bovine sperm and pig ASC was performed. The experimental protocol is shown below.
(i) Porcine ASC was cultured with pig sperm in a sheet-like or floating state (co-culture of ASC and sperm).
(ii) Use PBS (-) or DMEM / Ham's F12 medium (indicated as "medium" in the figure) supplemented with 0.1% HSA as the culture medium. In the control group, only porcine sperm floats in these. And cultured. In the co-culture group, pig adipose stem cells (designated as “ASC.sus” in the figure) and pig spermatozoa in sheet form (designated as “ASC” in the figure) or suspended were co-cultured.
(iii) After culturing, remove spermatozoa at 0 hours (control group only) and 5 hours later, place them in a hemocytometer (C-Chip), observe their motility with an inverted phase-contrast microscope, and observe sperm movement. Shot for 20 seconds.
(iv) Analyzing the movement of the photographed sperm, the ratio of the moving sperm to the total number of sperm (moment rate,% Motile spermatozoa) and the ratio of the number of moving sperm to the number of moving sperm (Advance rate,% Progressively motile spermatozoa) was calculated.
(v) The result of repeating twice was shown by the average value +/- standard error. After analyzing the effect of treatment and incubation time on the motility rate and forward motility rate using a two-way analysis of variance, a significant difference was found in the interaction. The test was conducted by Tukey's multiple comparison test.
（２）結果
準備実験として牛凍結融解精子とブタ培養ASCシート（ロンザ社）を共培養した結果、共培養1時間の時点において、運動する精子の割合（運動率）が約50％であり、共培養していない場合の運動率25％よりも上昇していた。培養を継続したところ、培養2時間目から、共培養の有無によって、精子の直進運動性が異なる傾向を認めた。培養22時間後の評価では、共培養の有無に関わらず精子の運動率は約1％であったが、ASCと共培養した精子において、共培養しなかった場合には全く認められなかった活発に前進運動する精子の存在が確認された。即ち、ブタASCが精子の前進運動性を高める又は長時間維持する効果を有することが示唆された。
(2) Results As a result of co-culturing bovine freeze-thawed sperm and swine cultured ASC sheet (Lonza) as a preparatory experiment, the proportion of sperm to move (motility rate) is about 50% at 1 hour of co-culture. The motility rate was higher than 25% without co-culture. When culturing was continued, from the 2nd hour of culturing, the motility of spermatozoa tended to differ depending on the presence or absence of co-culture. In the evaluation after 22 hours of culturing, the sperm motility rate was about 1% regardless of the presence or absence of co-culture, but in sperm co-cultured with ASC, no activity was observed when co-culture was not performed. The presence of sperm moving forward was confirmed. That is, it was suggested that porcine ASC has an effect of enhancing the forward motility of sperm or maintaining it for a long time.
本実験では、ブタ精子とブタASCを共培養し、その効果を調べた。6〜8時間の共培養の結果、共培養しない場合又は浮遊状態のブタASCとブタ精子を混和して培養した場合に比べ、精子の運動性は高く維持されただけでなく、精子の受精能力として重要である前進運動する精子の割合が上昇した（図１、図２）。確認の目的でブタASC培養上清中のサイトカイン濃度を測定したが、培養前後で差はなかった。 In this experiment, porcine sperm and porcine ASC were co-cultured and their effects were examined. As a result of co-culturing for 6 to 8 hours, sperm motility was not only maintained higher than when culturing without culturing or mixing swine ASC and swine sperm, but also fertilizing ability of sperm As a result, the proportion of sperm that moves forward is important (FIGS. 1 and 2). For confirmation, the cytokine concentration in the porcine ASC culture supernatant was measured, but there was no difference between before and after the culture.
次いで、ブタの精子をヒトASCシート（ロンザ社）と共培養した。その結果、培養4時間で共培養しない場合に比べて、共培養した場合において精子の前進運動性が高いことが確認された。左精索静脈瘤症例のヒト精子をヒトASCシート（ロンザ社）と共培養した結果、共培養2時間で精子の運動率に差が出始め、ヒト精子採取後21.5時間（培養開始後16時間）においては、コントロール群（精子のみを培養）の精子が全く運動を示さなかった（運動率0％）のに対して、共培養群の運動率は55％（目視による）であった。
The pig sperm were then co-cultured with human ASC sheets (Lonza). As a result, it was confirmed that the forward motility of spermatozoa was higher when co-cultured than when co-culture was not performed after 4 hours of culture. As a result of co-culture of human sperm from a case of left varicocele with human ASC sheet (Lonza), the difference in sperm motility began in 2 hours of co-culture, 21.5 hours after collection of human sperm (16 hours after the start of culture) ), The sperm of the control group (cultured only sperm) showed no movement (
以上の通り、以下の組み合わせで精子とASCを共培養し、ASCの精子活性化現象を詳細に確認した。
（ａ）ブタ精子とブタASC（シート培養と混和培養）
（ｂ）ブタ精子とヒトASCシート
（ｃ）ヒト精子とヒトASCシート
As described above, sperm and ASC were co-cultured in the following combinations, and the sperm activation phenomenon of ASC was confirmed in detail.
(A) Pig sperm and pig ASC (sheet culture and mixed culture)
(B) Pig sperm and human ASC sheet (c) Human sperm and human ASC sheet
以上一連の実験結果より、１）ASCは精子の運動率を高く維持し、且つ精子の前進運動性を活性化する又は高率に維持すること、２）ASCの精子活性化作用は、分泌されるサイトカインが介在するのではなく、精子とASCの接触によること、及び３）精子とASCの共培養による効果（精子活性化）は種を超えて認められること、が判明した。この現象、即ちASCによる精子活性化は、ヒト静脈瘤による男子不妊症の細胞治療として応用可能であり、精子運動率低下の病態（例えば、ブタの夏期不妊症）に対して、種を超えて有用な治療技術と考えられる。尚、作用機序に関して、液性因子すなわちサイトカインの関与がないことと、細胞接触で運動性が高まった事実から、精子がASCに短期間接触すること、すなわち細胞間相互作用がtouch and goで行わることによって効果が発現しているものと推測される。 From the above series of experimental results, 1) ASC maintains a high sperm motility rate and activates or maintains a high rate of sperm forward motility. 2) The sperm activation action of ASC is secreted. It was found that the effect of the co-culture of sperm and ASC (sperm activation) is recognized across species, not by the cytokines involved. This phenomenon, ie, sperm activation by ASC, can be applied as a cell therapy for male infertility due to human varicose veins, and it is beyond species for pathological conditions of reduced sperm motility (eg, summer infertility in pigs) It is considered a useful therapeutic technique. Regarding the mechanism of action, the fact that humoral factors or cytokines are not involved, and the fact that motility was increased by cell contact, sperm contacted ASC for a short period of time, that is, cell-cell interaction was touch and go. It is presumed that the effect is manifested by performing.
２．各種細胞の精子活性化効果
ASCと精子を共培養することにより精子の運動性が高く維持されることが示された（上記実験）。更なる検討として、この精子活性化効果がASCに特異的であるか否かを調べることにした。具体的には、種々の異なるタイプの細胞とブタ精子を共培養し、その効果を検討することにした。また、セルインサートを使用することにより、ASCと精子が接触した培養環境と非接触の培養環境を作り出すことによって、細胞と精子の接触が精子の活性化を引き起こすことの実証を試みた。
2. Sperm activation effect of various cells
It was shown that the co-culture of ASC and sperm maintains high sperm motility (the above experiment). As a further study, we decided to investigate whether this sperm activation effect is specific to ASC. Specifically, we decided to co-cultivate various different types of cells and porcine spermatozoa and examine their effects. In addition, by using cell inserts, we tried to demonstrate that contact between cells and sperm causes sperm activation by creating a culture environment where ASC and sperm are in contact with each other and a non-contact culture environment.
（１）材料と方法
ヒト脂肪組織由来幹細胞（ASC）、ヒト骨髄由来幹細胞（BM-MSC）、ヒト歯髄由来幹細胞（DPSC）およびヒト皮膚由来線維芽細胞（NHDF: Normal-Human-Dermal-Fibroblasts)）をそれぞれ常法に従い24ウェル培養プレートでコンフルエント状態になるまで培養した。また、セルインサート（ポアサイズ1.0μm、メンブレンとウェル底面までの距離0.8 mm）のメンブレン底面にASCを接着させたのち、24ウェルプレートのウェルへ装着した。コントロールとしてウェル底面でASCを培養した後、セルインサート（裏面へのASCの接着なし）をウェルに装着したものを用意した。精子との培養直前に培地を0.3%ヒト血清アルブミン添加DMEM/Ham’s F12培養液へ交換した。精子と共培養した細胞の数は各細胞1.0〜1.5 x 105/ウェル/500μlであった。
(1) Materials and methods Human adipose tissue-derived stem cells (ASC), human bone marrow-derived stem cells (BM-MSC), human dental pulp-derived stem cells (DPSC), and human skin-derived fibroblasts (NHDF: Normal-Human-Dermal-Fibroblasts) ) Were each cultured in a 24-well culture plate according to a conventional method until confluent. In addition, ASC was adhered to the bottom surface of the cell insert (pore size: 1.0 μm, distance between membrane and bottom surface of 0.8 mm), and then attached to the well of a 24-well plate. A control was prepared by culturing ASC on the bottom of the well and then mounting the cell insert (no ASC adhered to the back) on the well. Immediately before culturing with sperm, the medium was replaced with 0.3% human serum albumin-added DMEM / Ham's F12 medium. The number of cells co-cultured with sperm was 1.0-1.5 × 10 5 / well / 500 μl of each cell.
手圧法にて採取した濃厚部ブタ精液をBTS希釈液にて希釈後、17℃にて使用直前まで保存した。使用に際して保存精子を洗浄した後、最終添加精子濃度が2.0 x 106/mlとなるように調整し、上記の通り準備したウェルへ添加して各細胞との共培養を開始した。また、セルインサートの入ったウェルにおいてはセルインサートの中へ精子を添加して培養を開始した。全ての細胞について、培養条件は37.0℃、5%CO2/95%空気の気相条件とした。 The concentrated porcine semen collected by the manual pressure method was diluted with a BTS diluent and stored at 17 ° C. until just before use. After use, the preserved sperm was washed, adjusted to a final added sperm concentration of 2.0 × 10 6 / ml, added to the well prepared as described above, and co-culture with each cell was started. Moreover, in the well containing the cell insert, sperm was added to the cell insert and the culture was started. For all cells, the culture conditions were gas phase conditions of 37.0 ° C. and 5% CO 2 /95% air.
培養終了後、直接（フラスコ培養の場合）あるいは培養した一部の精子をとり（ウェル培養の場合）、血球計算盤へ入れて精子を観察し、その運動性を21〜27秒間撮影した。撮影した画像を解析し、目視にて運動率（％）を判定するとともに、撮影した映像に現れた前進運動精子の実数をカウントした。 After completion of the culture, a part of the cultured sperm was taken directly (in the case of flask culture) or in the case of a well culture, put into a hemocytometer, and the sperm was observed, and its motility was photographed for 21 to 27 seconds. The captured images were analyzed, and the rate of movement (%) was visually determined. The number of forward moving spermatozoa that appeared in the captured images was counted.
（２）結果
＜異なる細胞タイプとの共培養（7時間）がブタ精子の運動率へ及ぼす影響＞
培養フラスコ内でコンフルエント状態となった各細胞上で精子を7時間培養した。その結果、コントロール群（精子単独の培養）では運動率が約1％であったのに対して、各細胞（ASC、BM-MSC、DPSC又はNHDF）との共培養では運動率が5〜10％であり、いずれも運動率が高い傾向を示した（図３）。
(2) Results <Effect of co-culture with different cell types (7 hours) on porcine sperm motility>
Sperm were cultured for 7 hours on each confluent cell in the culture flask. As a result, the motility rate was about 1% in the control group (culture of sperm alone), whereas the motility rate was 5-10 in the co-culture with each cell (ASC, BM-MSC, DPSC or NHDF). %, Both showed a tendency to have a high exercise rate (FIG. 3).
＜細胞と精子が接触しないウェルと接触するウェルにおけるブタ精子活性化の比較＞
ウェルの底面でASCを培養した後、セルインサート（裏面へのASCの接着なし）をウェルに装着し、セルインサート内でブタ精子を共培養した場合（セルインサートで隔離されるため、精子はASCに接触できない）に比べ、セルインサートの裏面にASCを接着させた後、セルインサート内でブタ精子を共培養した場合（セルインサートの膜を介して精子はASCに接触可能）は、培養5時間後の精子運動率が高く維持されていた（図４）。また、培養7時間後において、前進運動を示す精子の数（精子の運動を約20秒間撮影した映像中でカウントされた前進運動精子の実数）も同様に、細胞と精子が接触できた場合の方が格段に多い（図５）。尚、尾部の振動はASCとの接触があった精子の方が活発であり、接触のなかった運動精子では尾部の振動は極めて微弱であった。
<Comparison of porcine sperm activation in wells in contact with wells where cells and sperm do not contact>
After culturing ASC on the bottom of the well, when a cell insert (no ASC adheres to the back) is attached to the well and swine sperm is co-cultured in the cell insert (because it is isolated by the cell insert, the sperm is
（３）考察
いずれの細胞を用いても、コントロール群に比べ、共培養した場合に精子の運動性が高い傾向を示した。この結果は、ASCのみならず一般に他の体細胞との接触が精子の運動性を維持する可能性を示唆する。また、精子とASCが接触した状態で共培養した場合において、非接触の共培養より精子の運動率が高く、且つ前進運動性精子数が多かったことから、細胞と精子の接触が精子の運動性の維持ないし上昇（即ち、活性化）に重要であるといえる。
(3) Discussion When any of the cells was used, sperm motility tended to be higher when co-cultured than in the control group. This result suggests that contact with not only ASC but generally other somatic cells may maintain sperm motility. In addition, when co-cultured in a state where sperm and ASC were in contact, the sperm motility rate was higher than that of non-contact co-culture, and the number of forward motility spermatozoa was higher. It can be said that it is important for maintaining or increasing sex (that is, activation).
３．体外受精実験
（１）材料と方法
（ａ）卵子
と場にて未成熟雌ブタより卵巣を採取し、ペニシリンナトリウム60μg/ml及び硫酸ストレプトマイシン100μg/mlを添加した滅菌生理食塩水（35℃）に入れ、実験室まで持ち帰った。同様の滅菌生理食塩水で卵巣を洗浄後、卵巣表面の直径3〜6 mmの卵胞より卵子を吸引採取した。採取した卵胞を10% ウシ胎子血清（FCS）を添加した20 mM Hepes緩衝 M199培養液（ペニシリンナトリウム60μg/ml及び硫酸ストレプトマイシン100μg/ml添加）で4回洗浄した後、修正したNCSU37培養液（mNCSU37培養液；0.6 mM システイン、1 mM dibutyryl cyclic AMP、10 iu/ml eCG、10 iu/ml hCG、ペンシリンナトリウム60μg/ml、硫酸ストレプトマイシン100μg/ml及び10% ブタ卵胞液を添加）で4回洗浄した。洗浄後、同培養液中にて、38.5℃、5%CO2/95%空気の気相下で24時間培養した。dibutyryl cAMP、eCG及びhCGを含まないmNCSU37培養液で4回洗浄後、同培養液中でさらに24時間培養した。
3. In vitro fertilization experiment (1) Materials and methods (a) Eggs and ovaries were collected from immature sows in place and placed in sterile physiological saline (35 ° C) supplemented with 60 μg / ml penicillin sodium and 100 μg / ml streptomycin sulfate. I took it back to the laboratory. After washing the ovary with the same sterile physiological saline, the ovum was aspirated and collected from a follicle having a diameter of 3 to 6 mm on the surface of the ovary. The collected follicles were washed 4 times with 20 mM Hepes buffered M199 medium supplemented with 10% fetal calf serum (FCS) (60 μg / ml penicillin sodium and 100 μg / ml streptomycin sulfate added), and then modified NCSU37 medium (mNCSU37 Washed 4 times with culture solution; 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP, 10 iu / ml eCG, 10 iu / ml hCG, pencillin sodium 60 μg / ml,
（ｂ）精子の前培養
用手法にて採取した大ヨークシャー種雄豚の濃厚部精液をBTS希釈液（50 μg/ml 硫酸カナマイシン添加）で希釈後、17℃にて実験室まで運搬した。17℃にて使用まで保存後、2.5mlの希釈精液をとり、室温にて10分間放置することにより細胞塊を沈殿させた。上清 1.5 ml を 0.1% PVA 添加スクロース培地に重層し、120g、5分間、続いて270g、7分間遠心した後、上清を除去した。精子前培養用培地、すなわち修正 M199 培養液（M199 ＋10％FCS ＋2.92 mM 乳酸カルシウム ＋0.91 mM Na・pyruvate +3.05 mM Glucose +penicillin +streptomycin, pH 7.8）で洗浄後、同培養液に 1 x 107 精子/mlあるいは 10 x 107 精子/mlとなるように再浮遊し、4時間前培養した。この時、それぞれ脂肪幹細胞を 3 x 106 cells/ml となるように添加あるいは無添加（コントロール）した。コントロールとして前培養用培地のみ添加した。
(B) Pre-culture of spermatozoa The concentrated semen of large Yorkshire boars collected by the method for sperm was diluted with BTS diluent (50 μg / ml kanamycin sulfate added) and then transported to the laboratory at 17 ° C. After storage at 17 ° C. until use, 2.5 ml of diluted semen was taken and allowed to stand at room temperature for 10 minutes to precipitate the cell mass. 1.5 ml of the supernatant was layered on sucrose medium supplemented with 0.1% PVA, centrifuged at 120 g for 5 minutes, then 270 g for 7 minutes, and then the supernatant was removed. After washing with sperm preculture medium, modified M199 medium (M199 + 10% FCS +2.92 mM calcium lactate +0.91 mM Na · pyruvate +3.05 mM Glucose + penicillin + streptomycin, pH 7.8) The cells were resuspended to x 10 7 sperm / ml or 10 x 10 7 sperm / ml and pre-cultured for 4 hours. At this time, adipose stem cells were added or not added (control) at 3 × 10 6 cells / ml. As a control, only the preculture medium was added.
（ｃ）脂肪組織幹細胞（ASC）
コンフルエント状のブタ脂肪由来幹細胞（約 1 x 106 cells）をトリプシン処理により剥離し、PBSで1回、次いで精子前培養用培地で1回洗浄後、同培養液に再浮遊し、精子の前培養に添加した（3 x 106 cells/ml）。
(C) Adipose tissue stem cells (ASC)
Confluent porcine adipose-derived stem cells (approximately 1 x 10 6 cells) are detached by trypsin treatment, washed once with PBS, then once with sperm preculture medium, and then resuspended in the same culture medium before sperm Added to culture (3 × 10 6 cells / ml).
（ｄ）体外受精
成熟培養終了後、卵子を受精培地（2 mM カフェイン，8 mM CaCl2 および 10 mg/ml BSA を含む修正合成卵管液；mSOF）で4回洗浄し、同培養液のマイクロドロップ内（90 μl）に入れた。前培養後の精子と脂肪由来幹細胞の両者を含む培養液を 10 μl 卵子（90 μl）へ添加することにより授精を行った（最終精子濃度 1 x 106/mlあるいは 10 x 106 精子/ml; 最終カフェイン濃度 2 mM; 最終脂肪幹細胞濃度 3 x 105 cells/ml）。12 時間同様の気相下で培養後，酢酸エタノール（1：3）で卵子を固定した。65時間固定後アセトオルセイン染色を行った後、精子の侵入と核相の判定を行った．判定は以下の通り行った。
（ｉ）精子の侵入率
精子が1つ以上侵入した卵子の割合
（ｉｉ）多精子受精率
精子が2つ以上侵入した卵子の割合
（iii）侵入した平均精子数
受精卵当たり侵入した精子の平均値
（ｉｖ）雄性前核形成率
雄性前核が認められる卵子の割合
（ｖ）正常受精率
多精子侵入ではない正常に受精した卵子の割合（雌性前核と侵入精子、雌雄両前核形成）
(D) After completion of in vitro fertilization maturation culture, the ovum is washed 4 times with a fertilization medium (modified synthetic fallopian tube solution containing 2 mM caffeine, 8 mM CaCl 2 and 10 mg / ml BSA; mSOF). Placed in microdrop (90 μl). Fertilization was performed by adding medium containing both pre-cultured sperm and adipose-derived stem cells to 10 μl eggs (90 μl) (final sperm concentration 1 x 10 6 / ml or 10 x 10 6 sperm / ml Final caffeine concentration 2 mM; final adipose stem cell concentration 3 x 10 5 cells / ml). After culturing in the same gas phase for 12 hours, the eggs were fixed with ethanol acetate (1: 3). After fixing for 65 hours, acetoorcein staining was performed, and sperm invasion and nuclear phase were determined. The determination was performed as follows.
(I) Sperm penetration rate Percentage of sperm invaded by one or more sperm (ii) Multisperm fertilization rate Percentage of sperm invaded by two or more sperm (iii) Average number of sperm invaded Average sperm invaded per fertilized egg Value (iv) Male pronucleus formation rate Percentage of eggs with male pronucleus observed (v) Normal fertilization rate Percentage of normally fertilized eggs that are not multisperm invaders (formation of female pronucleus and invading sperm, both male and female pronuclei)
（２）結果
（２−１）体外受精に使用した精子の運動性について
保存容器から取り出した直後の精子の運動率は約85％程度であったが、その運動はごく微弱な尾部の振動のみであり、活発な前進運動は全く見られなかった。精子を洗浄後、前培養用培地へ浮遊したところ一定の割合の活発な運動が確認されたが、前培養終了後は、コントロールの精子はほとんど運動していなかった。一方、ASCを添加して前培養した精子は25％程度前培養終了後に運動していた。
(2) Results (2-1) Sperm motility used for in vitro fertilization The sperm motility immediately after removal from the storage container was about 85%, but the movement was only a very weak tail vibration. No active forward movement was observed. After washing the sperm and floating on the preculture medium, a certain percentage of active movement was confirmed, but the control sperm hardly moved after the preculture. On the other hand, spermatozoa pre-cultured with ASC added moved about 25% after pre-culture.
（２−２）体外受精の結果について
結果を図６〜９に示す。図６の表にまとめたとおり、精子濃度1x106/mlで受精した場合、ASC非存在下で前培養した精子は全く卵子に侵入しなかったのに対し、ASC存在下で前培養した精子は82%の割合で卵子に侵入した。侵入した卵の多精子侵入率は93%であり、極めて高率に多精子侵入が見られた。雄性前核形成率は57％であったが、正常受精率は0％であった。
(2-2) Results of in vitro fertilization The results are shown in FIGS. As summarized in the table of FIG. 6, when fertilized at a sperm concentration of 1 × 10 6 / ml, sperm pre-cultured in the absence of ASC did not enter the egg at all, whereas sperm pre-cultured in the presence of
10x106/mlの精子濃度で授精させた場合、コントロールでは全く精子の侵入が認められなかったのに対し、ASC存在下で前培養した精子では14％の卵子に侵入が認められた（図６）。この受精卵は1個であり、雌雄両前核を有する正常受精卵であった（図８）。 When fertilized at a sperm concentration of 10 × 10 6 / ml, no sperm invasion was observed in the control, whereas sperm pre-cultured in the presence of ASC showed invasion in 14% of the eggs (FIG. 6). ). This fertilized egg was one and was a normal fertilized egg having both male and female pronuclei (FIG. 8).
（３）考察
前培養で脂肪組織由来幹細胞を添加することにより、運動性の低下した精子が受精不可能な状態から受精可能な状態になった。即ち、脂肪組織由来幹細胞が精子の運動性を劇的に改善し、受精を可能にした。
(3) Discussion By adding adipose tissue-derived stem cells in the pre-culture, sperm with reduced motility has changed from a state incapable of fertilization to a state in which fertilization is possible. That is, adipose tissue-derived stem cells dramatically improved sperm motility and enabled fertilization.
本発明の方法によれば精子の活性を高めることができる。本発明は例えば男子不妊症の治療・改善に利用できる。また、人工受精（成功率や効率の向上）、家畜の繁殖（人工授精の成功率や効率の向上）、育種・種の維持（例えば絶滅危惧種の維持、ペットの系統の維持又は交雑）等への利用も図られる。 According to the method of the present invention, sperm activity can be increased. The present invention can be used, for example, for the treatment and improvement of male infertility. Also, artificial fertilization (improving success rate and efficiency), breeding livestock (improving success rate and efficiency of artificial insemination), breeding / species maintenance (eg maintaining endangered species, maintaining or crossing pet lines), etc. It can also be used for.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (16)
（１）脂肪組織由来幹細胞、骨髄由来幹細胞、歯髄由来幹細胞及び線維芽細胞からなる群より選択される細胞と、精子とを共培養するステップ。 A sperm activation method comprising the following step (1):
(1) A step of co-culturing a sperm with a cell selected from the group consisting of adipose tissue-derived stem cells, bone marrow-derived stem cells, dental pulp-derived stem cells and fibroblasts.
（２）共培養後の精子を回収するステップ。 The method according to any one of claims 1 to 3, further comprising the following step (2):
(2) A step of collecting sperm after co-culture.
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| JP2016007161A (en) * | 2014-06-24 | 2016-01-18 | 国立大学法人名古屋大学 | Ova activation method and use thereof |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |