JP5852499B2 - Pre-freezing device for biological samples - Google Patents

Pre-freezing device for biological samples Download PDF

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JP5852499B2
JP5852499B2 JP2012093493A JP2012093493A JP5852499B2 JP 5852499 B2 JP5852499 B2 JP 5852499B2 JP 2012093493 A JP2012093493 A JP 2012093493A JP 2012093493 A JP2012093493 A JP 2012093493A JP 5852499 B2 JP5852499 B2 JP 5852499B2
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holding member
sample container
guide rod
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container holder
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滋弘 吉村
滋弘 吉村
武内　雅弘
雅弘 武内
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Taiyo Nippon Sanso Corp
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Description

本発明は、生体試料が保存された生体容器を液体窒素中に浸漬して生体試料を凍結する、生体試料の予備凍結装置に関する。   The present invention relates to a biological sample preliminary freezing apparatus for immersing a biological container in which a biological sample is stored in liquid nitrogen to freeze the biological sample.

従来より畜産業界や生殖医療等の分野において、細胞の凍結保存が広く行われている。プログラムフリーザー中で−１℃／分程度の冷却速度で凍結するような方法は、一般に緩慢凍結法と呼ばれ、研究室や細胞バンクなどで良く用いられている。例えば特許文献１には、冷却過程での生体試料生存率の向上を図ることを目的として、ストロー状生体試料封入容器をほぼ氷結状態とし、冷媒ガス中で所定の温度まで下げ、次いで冷媒液化ガス中で目的の温度まで下げる凍結方法が開示されている。特許文献２には、Ｎ−メチルアセトアミドを含む生殖細胞の凍結保存剤を用い、***の入ったストローを蓋をして液体窒素蒸気中で３０分静置し、続いて、液体窒素保管容器中に移して保存することが開示されている。特許文献３には、***の代謝速度を下げる十分遅い速度で、３段階の温度で凍結することが開示されている。
一方、ヒトＥＳ細胞やｉＰＳ細胞、受精卵などある種の細胞では凍結の際の細胞内外の氷晶形成によるダメージが無視できず解凍後の生存率が非常に低くなるため、細胞入り容器を液体窒素中に直接浸漬することにより凍結を行うことがある。例えば特許文献４や非特許文献１には、試料容器ごと液体窒素に直接浸漬し細胞を急速凍結させることが記載されている。 Conventionally, cryopreservation of cells has been widely performed in fields such as the livestock industry and reproductive medicine. A method of freezing in a program freezer at a cooling rate of about −1 ° C./min is generally called a slow freezing method and is often used in laboratories and cell banks. For example, in Patent Document 1, for the purpose of improving the survival rate of the biological sample in the cooling process, the straw-shaped biological sample enclosure is almost frozen, lowered to a predetermined temperature in the refrigerant gas, and then the refrigerant liquefied gas Among them, a freezing method for lowering to a target temperature is disclosed. In Patent Document 2, a germ cell cryopreservation agent containing N-methylacetamide is used, a straw containing semen is covered and left in liquid nitrogen vapor for 30 minutes, and then in a liquid nitrogen storage container. And storing it. Patent Document 3 discloses freezing at three stages of temperature at a sufficiently slow rate to lower the sperm metabolic rate.
On the other hand, in some types of cells such as human ES cells, iPS cells, and fertilized eggs, damage due to ice crystal formation inside and outside the cells during freezing cannot be ignored and the survival rate after thawing becomes very low. Freezing may be performed by direct immersion in nitrogen. For example, Patent Document 4 and Non-Patent Document 1 describe that a sample container is directly immersed in liquid nitrogen to rapidly freeze cells.

特開平５−２８５１６３号公報JP-A-5-285163 特開平１１−２２８３０１号公報JP 11-228301 A 特開２００４−５０５６２４号公報JP 2004-505624 A 特開２０１０−２７３５４９号公報JP 2010-273549 A

凍結保存、編集者：酒井昭、朝倉書店発行、１９８７年５月２５日発行、ｐ９Cryopreservation, Editor: Akira Sakai, Asakura Shoten, May 25, 1987, p9

ところで、液体窒素中への直接浸漬を実施する場合、容器のヘッド部を作業者が指やピンセット等の工具で摘まんで液体窒素に浸漬し、保持する方法を取っている。この場合、容器内の試料が確実に凍結され、且つキャップ部分からの液体窒素の混入がないよう、容器に対する液体窒素液面の位置を一定に保つ必要がある。しかしながら、液面位置を目視で確認しながらの作業であるため、凍結処理の成否は作業者の技量に大きく依存し、安定した凍結処理が困難であった。また、液体窒素の液面に作業者の手指が接近するため凍傷のリスクもある。   By the way, when direct immersion in liquid nitrogen is performed, a method is adopted in which an operator picks the head portion of the container with a tool such as a finger or tweezers, soaks it in liquid nitrogen, and holds it. In this case, it is necessary to keep the position of the liquid nitrogen liquid surface relative to the container constant so that the sample in the container is reliably frozen and liquid nitrogen is not mixed from the cap portion. However, since the operation is performed while visually confirming the liquid level position, the success or failure of the freezing process greatly depends on the skill of the operator, and a stable freezing process is difficult. There is also a risk of frostbite because the operator's fingers approach the liquid nitrogen surface.

本発明は、このような実情に鑑みてなされたものであり、試料容器の液体窒素への直接浸漬を安全に行うことができ、作業者の技量に依存することなく生体試料の急速凍結処理を安定に行うことが可能な生体試料の予備凍結装置を提供することを目的とする。   The present invention has been made in view of such circumstances, and can directly immerse a sample container in liquid nitrogen safely, and can perform rapid freezing treatment of a biological sample without depending on the skill of the operator. An object of the present invention is to provide a pre-freezing device for a biological sample that can be stably performed.

本発明者らは、前記実情に鑑みてなされたものであり、生体試料を保持部材に保持した、試料容器ホルダーをガイドロッドに保持された状態で液体窒素中に浸漬する際に液体窒素液面近傍に浮遊可能な装置を用いることにより上記課題が解決できることを見出し、本発明を完成させるに至った。   The inventors of the present invention have been made in view of the above circumstances, and the liquid nitrogen liquid level is obtained when the biological sample is held in the holding member and the sample container holder is immersed in liquid nitrogen while being held by the guide rod. It has been found that the above problem can be solved by using a device capable of floating in the vicinity, and the present invention has been completed.

すなわち本発明は、以下の（１）〜（６）に記載する発明を要旨とする。
（１）液体窒素液面近傍に浮遊可能な、試料容器ホルダーと、それを保持するガイドロッドとを備えた、試料容器を液体窒素に直接浸漬させて試料容器内の生体試料を凍結するための予備凍結装置であって、
（ｉ）試料容器ホルダーには試料容器を保持する保持部材が設けられており、
（ii）保持部材の一部もしくは全部が液体窒素に浮遊可能な部材から形成され、
（iii）保持部材には、ガイドロッドを貫通させるための貫通孔が設けられており、
（iv）ガイドロッドの下端には試料容器ホルダーの落下防止用ストッパーが設けられていて、
（ｖ）ガイドロッドが保持部材の貫通孔を貫通して配置されることにより、試料容器ホルダーをストッパーより上側で移動可能に支持している
ことを特徴とする、生体試料の予備凍結装置。 That is, the gist of the present invention is the invention described in the following (1) to (6).
(1) A sample container holder that can float in the vicinity of the liquid nitrogen liquid surface and a guide rod that holds the sample container holder, and for freezing a biological sample in the sample container by directly immersing the sample container in liquid nitrogen A pre-freezing device,
(I) The sample container holder is provided with a holding member for holding the sample container,
(Ii) a part or all of the holding member is formed of a member that can float in liquid nitrogen;
(Iii) The holding member is provided with a through hole for allowing the guide rod to pass therethrough,
(Iv) A stopper for preventing the sample container holder from falling is provided at the lower end of the guide rod,
(V) The biological sample preliminary freezing apparatus, wherein the guide rod is disposed through the through hole of the holding member to support the sample container holder so as to be movable above the stopper.

（２）前記試料容器ホルダーが、試料容器を保持するための保持部材として、１以上の試料容器を貫通して保持するための挿入孔を有する上側保持部材、試料容器の底部を支持する下側保持部材、及び上側保持部材と下側保持部材とを連結する１又は２以上の固定支柱を備え、
前記上側保持部材および下側保持部材の相対する位置にはガイドロッドを貫通させるための貫通孔がそれぞれ設けられており、
ガイドロッドが前記貫通孔を貫通することにより、試料容器ホルダーをストッパーより上側で移動可能な状態で支持している
ことを特徴とする、前記（１）に記載の生体試料の予備凍結装置。
（３）上側保持部材と下側保持部材のいずれか一方又は双方の一部もしくは全部が液体窒素に浮遊可能な部材で形成されていることを特徴とする、前記（２）に記載の生体試料の予備凍結装置。
（４）下側保持部材の下部側に浮力調節手段が配置されていることを特徴とする、前記（２）又は（３）に記載の生体試料の予備凍結装置。
（５）前記ガイドロッドがガラスエポキシ樹脂、ポリテトラフルオロエチレン樹脂、もしくはフェノール樹脂、又はこれらの樹脂の繊維強化複合材料であることを特徴とする、前記（１）〜（４）のいずれかに記載の生体試料の予備凍結装置。
（６）前記ガイドロッドの上端が自動昇降機に接続されていることを特徴とする、前記（１）〜（５）のいずれかに記載の生体試料の予備凍結装置。 (2) The sample container holder serves as a holding member for holding the sample container, an upper holding member having an insertion hole for penetrating and holding one or more sample containers, and a lower side for supporting the bottom of the sample container A holding member, and one or more fixed struts connecting the upper holding member and the lower holding member,
Through holes for penetrating the guide rods are respectively provided at the opposing positions of the upper holding member and the lower holding member,
The biological sample preliminary freezing apparatus according to (1) above, wherein the sample container holder is supported so as to be movable above the stopper by allowing the guide rod to pass through the through hole.
(3) The biological sample according to (2) above, wherein one or both of the upper holding member and the lower holding member are formed of a member capable of floating in liquid nitrogen. Preliminary freezing equipment.
(4) The biological sample preliminary freezing apparatus according to (2) or (3) above, wherein buoyancy adjusting means is disposed on the lower side of the lower holding member.
(5) The guide rod is a glass epoxy resin, polytetrafluoroethylene resin, phenol resin, or a fiber-reinforced composite material of these resins. The biological sample preliminary freezing apparatus as described.
(6) The biological sample preliminary freezing apparatus according to any one of (1) to (5), wherein an upper end of the guide rod is connected to an automatic elevator.

本発明の予備凍結装置によれば、試料容器ホルダーが液体窒素の液面近傍で浮遊することによって液体窒素の液面の上下動に試料容器ホルダーが追従するので、試料容器の液体窒素への浸漬状態がほぼ一定に保たれる。よって、作業者の技量によらず生体試料の安定な凍結が実現される。
また、ガイドロッドの上昇及び下降によって試料容器ホルダーごと試料容器の液体窒素への浸漬及び取り出しが速やかに行われるため、作業者の凍傷のリスクを低減し、且つ、作業の安定化を図ることもできる。
更に、自動昇降装置を用いて、ガイドロッド上端部分を自動昇降装置に接続する場合には作業者の凍傷をより確実に防止することが可能になる。 According to the preliminary freezing apparatus of the present invention, since the sample container holder follows the vertical movement of the liquid nitrogen liquid surface by floating near the liquid nitrogen liquid surface, the sample container is immersed in liquid nitrogen. The state is kept almost constant. Therefore, stable freezing of the biological sample is realized regardless of the skill of the operator.
Also, as the guide rod moves up and down, the sample container holder and the sample container are quickly immersed and removed from the liquid nitrogen, reducing the risk of frostbite for the operator and stabilizing the work. it can.
Furthermore, when the automatic lifting device is used to connect the upper end portion of the guide rod to the automatic lifting device, it becomes possible to prevent the frostbite of the worker more reliably.

図１は、本発明の予備凍結装置の一例を示す概念断面図である。FIG. 1 is a conceptual cross-sectional view showing an example of the preliminary freezing apparatus of the present invention. 図２は、本発明の予備凍結装置の他の例を示す概念断面図である。FIG. 2 is a conceptual cross-sectional view showing another example of the preliminary freezing apparatus of the present invention. 図３は、本発明の予備凍結装置のさらに他の例を示す概念断面図である。FIG. 3 is a conceptual cross-sectional view showing still another example of the preliminary freezing apparatus of the present invention. 図４は、本発明の自動昇降機付き予備凍結装置の概念図であり、試料容器ホルダーが液体窒素中に浸漬されている状態を示す概念図である。FIG. 4 is a conceptual diagram of a preliminary freezing apparatus with an automatic elevator according to the present invention, and is a conceptual diagram showing a state in which a sample container holder is immersed in liquid nitrogen. 図５は、本発明の自動昇降機付き予備凍結装置の概念図であり、試料容器ホルダーが液体窒素の液面上に持ち上げられている状態を示す概念図である。FIG. 5 is a conceptual diagram of the preliminary freezing apparatus with an automatic elevator according to the present invention, and is a conceptual diagram showing a state in which the sample container holder is lifted on the liquid nitrogen level.

本発明の「生体試料の予備凍結装置」は、液体窒素液面近傍に浮遊可能な、試料容器ホルダーと、それを保持するガイドロッドとを備えた、試料容器を液体窒素に直接浸漬させて試料容器内の生体試料を凍結するための予備凍結装置であって、
（ｉ）試料容器ホルダーには試料容器を保持する保持部材が設けられており、
（ii）保持部材の一部もしくは全部が液体窒素に浮遊可能な部材から形成され、
（iii）保持部材には、ガイドロッドを貫通させるための貫通孔が設けられており、
（iv）ガイドロッドの下端には試料容器ホルダーの落下防止用ストッパーが設けられていて、
（ｖ）ガイドロッドが保持部材の貫通孔を貫通して配置されることにより、試料容器ホルダーをストッパーより上側で移動可能に支持していることを特徴とする。 The “biological sample pre-freezing device” of the present invention comprises a sample container holder that can float near the liquid nitrogen liquid surface and a guide rod that holds the sample container holder. A pre-freezing device for freezing a biological sample in a container,
(I) The sample container holder is provided with a holding member for holding the sample container,
(Ii) a part or all of the holding member is formed of a member that can float in liquid nitrogen;
(Iii) The holding member is provided with a through hole for allowing the guide rod to pass therethrough,
(Iv) A stopper for preventing the sample container holder from falling is provided at the lower end of the guide rod,
(V) The guide rod is disposed so as to penetrate the through hole of the holding member, thereby supporting the sample container holder so as to be movable above the stopper.

以下、本発明の「生体試料の予備凍結装置」を図１〜５を用いて説明する。尚、これらの図は本発明の具体的態様の例示であり、本発明はこれらの図に示す予備凍結装置の例になんら限定されるものではない。
図１には、本発明の「生体試料の予備凍結装置」の典型例を示す。試料容器ホルダー１中の保持部材は、上側保持部材３と下側保持部材４とから形成されていて、固定支柱７で一定間隔に支持されている。試料容器５は、下側保持部材４によってその底面を支持されるとともに、上側保持部材３に設けられた挿入孔１１に差し込まれることによってその直立状態が保持される。
下側保持部材４を、図１に示すように、第１プレート部材１５と、該プレート部材に貼り合わされた第２プレート部材１６から形成された積層構造にすると、保持部材の製作を容易にすることができる。
また、上側保持部材３と下側保持部材４の略中央部にそれぞれ設けられた貫通孔１３、貫通孔１４には、所定のクリアランスをもってガイドロッド８が貫通している。ガイドロッド８の下端部には貫通孔１４の内径よりは外径のやや大きい部材からなるストッパー９が設けられ、ストッパー９より上側で試料容器ホルダー１がガイドロッド８の軸方向に移動可能に支持されている。 Hereinafter, the “biological sample preliminary freezing apparatus” of the present invention will be described with reference to FIGS. These drawings are illustrations of specific embodiments of the present invention, and the present invention is not limited to the examples of the preliminary freezing apparatus shown in these drawings.
FIG. 1 shows a typical example of the “biological sample preliminary freezing apparatus” of the present invention. The holding member in the sample container holder 1 is formed of an upper holding member 3 and a lower holding member 4 and is supported by fixed columns 7 at regular intervals. The bottom surface of the sample container 5 is supported by the lower holding member 4, and the sample container 5 is held in an upright state by being inserted into the insertion hole 11 provided in the upper holding member 3.
As shown in FIG. 1, when the lower holding member 4 has a laminated structure formed of a first plate member 15 and a second plate member 16 bonded to the plate member, the holding member can be easily manufactured. be able to.
Further, the guide rod 8 passes through the through hole 13 and the through hole 14 respectively provided at substantially the center portions of the upper holding member 3 and the lower holding member 4 with a predetermined clearance. A stopper 9 made of a member having an outer diameter slightly larger than the inner diameter of the through hole 14 is provided at the lower end of the guide rod 8, and the sample container holder 1 is supported above the stopper 9 so as to be movable in the axial direction of the guide rod 8. Has been.

図２は、予備凍結装置の他の例を示す図であり、保持部材２が試料容器５をキャップ６の下側面で保持する例である。保持部材２は、図１に示す第２プレート部材１６と同様に、第１プレート部材１７と、該プレート部材に貼り合わされた第２プレート部材１８から形成された積層構造になっている。
図３は、予備凍結装置の更に他の例を示す図であり、第１プレート部材１７と第２プレート部材１８から形成されている保持部材２が試料容器５の略下半分を保持している例である。
図４及び図５は、自動昇降機２３が配設された、図１に対応する予備凍結装置において、試料容器４が液体窒素２１に浸漬された状態、試料容器５が液体窒素２１の液面上に持ち上げられている状態をそれぞれ示している。 FIG. 2 is a diagram showing another example of the preliminary freezing apparatus, in which the holding member 2 holds the sample container 5 on the lower surface of the cap 6. Similar to the second plate member 16 shown in FIG. 1, the holding member 2 has a laminated structure formed of a first plate member 17 and a second plate member 18 bonded to the plate member.
FIG. 3 is a view showing still another example of the preliminary freezing apparatus, in which the holding member 2 formed of the first plate member 17 and the second plate member 18 holds the substantially lower half of the sample container 5. It is an example.
4 and FIG. 5 show a state in which the sample container 4 is immersed in the liquid nitrogen 21 in the preliminary freezing apparatus corresponding to FIG. Each state is shown as being lifted.

（１）保持部材
保持部材の材料は、常圧での液体窒素の温度に耐えるものであれば金属、樹脂等特に制限されるものではなく、液体窒素より比重の大きい材料及び小さい材料のいずれも使用可能であるが、塩化ビニル、ポリスチレン等の樹脂の使用が好ましく、これらの樹脂の中でも水分の吸収率の低い樹脂がより好ましい。
また、保持部材の一部もしくは全部が液体窒素より比重が小さく、液体窒素に浮遊可能な部材から形成されることが好ましい。該部材としては、発泡樹脂部材、中空部材等が挙げられる。保持部材内部へ侵入した水分が凍結することによる比重の変動を避けるため発泡樹脂部材の中でも独立気泡構造の発泡樹脂を使用することが好ましい。
尚、保持部材は、図２、３に示すように１つの形状物から形成することができ、図１に示すように上側保持部材と下側保持部材の２つの形状物から形成することができ、更に３つ以上の形状物から形成することもできる。
以下、図１〜図３に示す保持部材についてそれぞれ説明する。 (1) The material of the holding member holding member is not particularly limited as long as it can withstand the temperature of liquid nitrogen at normal pressure, and is not particularly limited. Although usable, it is preferable to use resins such as vinyl chloride and polystyrene, and among these resins, resins having a low moisture absorption rate are more preferable.
Further, it is preferable that a part or all of the holding member has a specific gravity smaller than that of liquid nitrogen and is formed of a member that can float on liquid nitrogen. Examples of the member include a foamed resin member and a hollow member. In order to avoid fluctuations in specific gravity due to freezing of moisture that has entered the holding member, it is preferable to use a foamed resin having a closed cell structure among the foamed resin members.
The holding member can be formed from one shape as shown in FIGS. 2 and 3, and can be formed from two shapes, an upper holding member and a lower holding member as shown in FIG. Further, it can be formed from three or more shapes.
Hereinafter, the holding members shown in FIGS. 1 to 3 will be described.

（１−１）図１に示す保持部材
（ｉ）上側保持部材
上側支持部材３には、図１に例示するように、円盤状のプレート部材に、試料容器５が挿入される挿入孔１１と、ガイドロッド８を貫通できる貫通孔１３が設けられている。
上側保持部材３の材料としては前述の材料が使用できる。上側保持部材３は、１枚のプレート形状物でもよく、また、複数枚を貼り合わせて形成することもできる。また、試料容器５の直立状態を実現可能であれば、フレームに適当な間隔で張り巡らしたワイヤで形成することもできる。 (1-1) The holding member shown in FIG. 1 (i) The upper holding member upper support member 3 includes an insertion hole 11 into which the sample container 5 is inserted into a disk-shaped plate member as illustrated in FIG. A through hole 13 that can penetrate the guide rod 8 is provided.
As the material of the upper holding member 3, the above-described materials can be used. The upper holding member 3 may be a single plate-shaped object, or may be formed by bonding a plurality of sheets. Further, if the upright state of the sample container 5 can be realized, the sample container 5 can be formed of a wire stretched around the frame at an appropriate interval.

（ii）下側保持部材
図１に示す下側保持部材４は、試料容器５が挿入される挿入孔１１及びガイドロッド貫通用の貫通孔１４を有する円盤状の第１プレート部材１５と、ガイドロッド貫通用の貫通孔１４を有する円盤状の第２プレート部材１６とを貼り合わせて一体化した積層構造物である。第１プレート部材１５及び第２プレート部材１６としては、前述の材料が使用でき、また第１プレート部材１５は、上側保持部材に用いた部材と同一のものを用いることができる。下側保持部材の一部もしくは全部は、液体窒素より比重が小さく液体窒素に浮遊可能な部材から形成されることが好ましい。
なお、下側保持部材は、２つの部材の貼り合わせた積層構造に限らず、１つの部材からなる構造、又は３つ以上の部材を貼り合わせた積層構造とすることもできる。 (Ii) Lower Holding Member A lower holding member 4 shown in FIG. 1 includes a disc-shaped first plate member 15 having an insertion hole 11 into which a sample container 5 is inserted and a through hole 14 for penetrating a guide rod, and a guide. It is a laminated structure in which a disk-shaped second plate member 16 having a through hole 14 for penetrating a rod is bonded and integrated. As the first plate member 15 and the second plate member 16, the above-described materials can be used, and the first plate member 15 can be the same as the member used for the upper holding member. Part or all of the lower holding member is preferably formed of a member having a specific gravity smaller than that of liquid nitrogen and capable of floating in liquid nitrogen.
Note that the lower holding member is not limited to a laminated structure in which two members are bonded together, and may have a structure including one member or a laminated structure in which three or more members are bonded together.

図１に示すように、試料容器ホルダー１において上側保持部材３と下側保持部材４の双方で試料容器５を保持することにより、上側保持部材３の挿入孔１１のクリアランスを大きめに設計でき、試料容器５の出し入れ作業が容易となる。また、さまざまな形状や寸法の試料容器５への対応が可能となり、汎用性が向上する。また、上下の２か所で試料容器５を支持するので、安定した液体窒素への浸漬が可能になる。   As shown in FIG. 1, by holding the sample container 5 by both the upper holding member 3 and the lower holding member 4 in the sample container holder 1, the clearance of the insertion hole 11 of the upper holding member 3 can be designed to be large, The sample container 5 can be taken in and out easily. Moreover, it becomes possible to deal with the sample containers 5 having various shapes and dimensions, and versatility is improved. In addition, since the sample container 5 is supported at the upper and lower portions, stable immersion in liquid nitrogen is possible.

（iii）固定支柱
試料容器ホルダー１は、固定支柱７を備える。図１に示すように、保持部材が上側保持部材３と下側保持部材１６からなる２つの部材から形成される場合には、これらの部材を固定支柱７で連結する。該固定支柱７の本数は任意であり、また、固定支柱７の長さは、使用する試料容器５の長さにより適宜決定することができる。固定支柱７の材料は機械的強度を維持でき、液体窒素温度程度の低温に耐えるものであれば特に制限されるものではないが、金属、樹脂等を用いることができ、軽量性の点から樹脂が好ましい。また、樹脂の中でも水分の吸収率の低い樹脂が好ましい。 (Iii) The fixed column sample container holder 1 includes a fixed column 7. As shown in FIG. 1, when the holding member is formed of two members including the upper holding member 3 and the lower holding member 16, these members are connected by a fixed column 7. The number of the fixed struts 7 is arbitrary, and the length of the fixed struts 7 can be appropriately determined depending on the length of the sample container 5 to be used. The material of the fixed support 7 is not particularly limited as long as it can maintain the mechanical strength and can withstand a low temperature of about liquid nitrogen temperature. However, metal, resin, etc. can be used, and resin from the viewpoint of light weight. Is preferred. Of the resins, a resin having a low moisture absorption rate is preferable.

（１−２）図２、３に示す保持部材
保持部材は、上側保持部材および下側保持部材の２つの部材から構成される場合に限らず、図２、図３に例示するように、１つの部材から構成してもよい。図２においては、試料容器５のキャップ６の外径より挿入孔１１の径を小さく設定することにより、試料容器５がキャップ６下側面で第１プレート部材１７に支持され、試料容器ホルダー１を上方に引き上げた際の落下を防止することが可能になる。
図３に示す保持部材において、試料容器５とほぼ同一形状の挿入孔１１を設け、試料容器５を挿入孔１１に押し込むように装着することにより、試料容器ホルダー１を液体窒素中に浮かべた際の試料容器５の脱落が防止される。 (1-2) The holding member holding member shown in FIGS. 2 and 3 is not limited to the case where the holding member holding member is composed of the upper holding member and the lower holding member, and as illustrated in FIGS. You may comprise from one member. In FIG. 2, by setting the diameter of the insertion hole 11 to be smaller than the outer diameter of the cap 6 of the sample container 5, the sample container 5 is supported by the first plate member 17 on the lower surface of the cap 6, and the sample container holder 1 is attached. It is possible to prevent a drop when the cover is pulled upward.
In the holding member shown in FIG. 3, when the sample container holder 1 is floated in liquid nitrogen by providing an insertion hole 11 having substantially the same shape as the sample container 5 and mounting the sample container 5 so as to be pushed into the insertion hole 11. The sample container 5 is prevented from falling off.

（２）浮力調節手段
試料容器ホルダー１を液体窒素中で所望の浮遊位置とするために、図示は省略するが、必要に応じて保持部材とは別に浮力調節手段を装着することにより、試料容器ホルダー１全体の比重（浮力）を調節する。尚、本発明において、浮力調節手段とは、保持部材に直接貼り合わされた形状物ではなく、保持部材とは独立の形状物をいう。
浮力調節手段の設置個所は任意であるが、図１に示す試料容器ホルダー１の場合、下側保持部材４の下部側とすることが望ましい。液体窒素の液面を試料容器５のキャップ６の下端近傍に制御することが可能となるとともに、ガイドロッド８からストッパー９を取り外すことにより浮力調節手段の装着を容易に行うことができ、試料容器の数等に応じた浮力調節を容易に行えるからである。また、試料容器５の出し入れに支障をきたすことがないからである。図２、３の場合、同様の理由から、保持部材２下部側とすることが望ましい。
浮力調節手段の形状は任意であり、例えばプレート状、ブロック状とすることができる。また、浮力調節手段の水平方向の外形形状は、保持部材２、又は下側保持部材４と略同一もしくは小さめであることが好ましい。水平方向にスペースが広がらず装置全体の小型化につながるからである。なお、浮力調節手段の外形形状がプレート状である場合、ガイドロッド８が貫通可能なよう適宜貫通孔を設けておく。
浮力調節手段としては、目的に応じて任意の材料を用いることができ、例えば液体窒素よりも比重の小さい発泡樹脂部材、中空部材等を使用することができる。尚、上記発泡樹脂部材としては、連続気泡の場合水分が内部に侵入し凍結による比重の変化を生じるため、独立気泡構造を有するものを使用することが好ましい。 (2) Buoyancy adjustment means Although the illustration is omitted in order to place the sample container holder 1 in a desired floating position in liquid nitrogen, the sample container can be attached by attaching a buoyancy adjustment means separately from the holding member as necessary. Adjust the specific gravity (buoyancy) of the entire holder 1. In the present invention, the buoyancy adjusting means is not a shape directly bonded to the holding member but a shape independent of the holding member.
The location of the buoyancy adjusting means is arbitrary, but in the case of the sample container holder 1 shown in FIG. The liquid surface of liquid nitrogen can be controlled in the vicinity of the lower end of the cap 6 of the sample container 5, and the buoyancy adjusting means can be easily attached by removing the stopper 9 from the guide rod 8. This is because buoyancy can be easily adjusted according to the number of Moreover, it is because there is no trouble in taking in and out of the sample container 5. In the case of FIGS. 2 and 3, it is desirable that the holding member 2 is on the lower side for the same reason.
The shape of the buoyancy adjusting means is arbitrary, and can be, for example, a plate shape or a block shape. Further, it is preferable that the horizontal shape of the buoyancy adjusting means is substantially the same as or slightly smaller than that of the holding member 2 or the lower holding member 4. This is because the space does not spread in the horizontal direction, which leads to downsizing of the entire apparatus. In addition, when the outer shape of the buoyancy adjusting means is a plate shape, a through hole is provided as appropriate so that the guide rod 8 can pass therethrough.
As the buoyancy adjusting means, any material can be used depending on the purpose. For example, a foamed resin member, a hollow member, or the like having a specific gravity smaller than that of liquid nitrogen can be used. As the above-mentioned foamed resin member, in the case of open cells, it is preferable to use a member having a closed cell structure because moisture enters the inside and changes in specific gravity due to freezing.

（３）ガイドロッド
ガイドロッド８は、金属や樹脂等、液体窒素の低温に耐久性がある材料を選択して使用することができるが、ガラスエポキシ樹脂、テフロン（登録商標）、ベークライト（登録商標）等は、液化窒素の冷熱による軸周囲への氷の付着が発生し難く、試料容器ホルダー１の上下方向への移動がスムーズに行われるため、連続使用の観点から好ましい。
また、ストッパー９は、ガイドロッド８に対して着脱自在とされ、ガイドロッド８に例示した材料で形成することができる。ストッパー９の水平方向の外形は、例えば図１、図２、３等に示す貫通孔１２、１４の内径より大とされる。 (3) Guide rod The guide rod 8 can be selected from materials that are durable at low temperatures of liquid nitrogen, such as metal and resin, but can be used as glass epoxy resin, Teflon (registered trademark), Bakelite (registered trademark). ) Etc. are preferable from the viewpoint of continuous use because ice hardly adheres to the periphery of the shaft due to the cold heat of liquefied nitrogen and the sample container holder 1 moves smoothly in the vertical direction.
The stopper 9 is detachable from the guide rod 8 and can be formed of the material exemplified for the guide rod 8. The outer shape of the stopper 9 in the horizontal direction is made larger than the inner diameters of the through holes 12 and 14 shown in FIGS.

（４）自動昇降装置
本発明で使用可能な自動昇降装置２３としては、図４、５に示すように、ガイドロッド８の上部先端を保持するアーム２４と、任意に設定した上端位置と下端位置との間でアーム２４を上下方向に移動させるためのモーター式駆動機構と、上昇・下降釦（図示略）とを有する昇降機とから構成される装置が挙げられる。このような自動昇降装置２３においては、下降釦を押すことによりアーム２４を下端位置まで下降させ、予め設定した時間保持した後、上昇釦を押し下げることによりアーム２４を上端位置まで上昇させるといった動作が行われる。
図５に示すように、アーム２４を上端に位置させた状態では、ガイドロッド８の下端部に配置されたストッパー９が下側保持部材１６の下面を支えることにより試料容器ホルダー１を吊り上げる。このときストッパー９が設置されていることにより試料容器ホルダー１の落下が防止されて、真空断熱槽内の液体窒素２１の液面上に保持される。この状態において、作業者による試料容器ホルダー１への試料容器５のセット、及び後述する凍結処理後の試料容器５の取り出しが行われる。 (4) Automatic Lifting Device As shown in FIGS. 4 and 5, the automatic lifting device 23 usable in the present invention includes an arm 24 that holds the upper end of the guide rod 8, and arbitrarily set upper and lower positions. A motor-type drive mechanism for moving the arm 24 in the up and down direction, and an elevator having an up / down button (not shown). In such an automatic lifting device 23, the arm 24 is lowered to the lower end position by pressing the lowering button, and after holding for a preset time, the arm 24 is raised to the upper end position by depressing the raising button. Done.
As shown in FIG. 5, in a state where the arm 24 is positioned at the upper end, the stopper 9 disposed at the lower end of the guide rod 8 supports the lower surface of the lower holding member 16 to lift the sample container holder 1. At this time, since the stopper 9 is installed, the sample container holder 1 is prevented from dropping and held on the liquid surface of the liquid nitrogen 21 in the vacuum heat insulating tank. In this state, the operator sets the sample container 5 in the sample container holder 1 and removes the sample container 5 after the freezing process described later.

一方、下降釦を押すことにより図４に示すように、アーム２４が下がった状態となる。試料容器ホルダー１がガイドロッド８のストッパー９よりも上部で自在に移動可能となっているため、液体窒素２１中に浮遊した状態となる。この状態において、試料容器ホルダー１にセットされた試料容器５が液体窒素２１に浸漬するため、凍結処理が行われる。
その後、上昇釦を押すことによりアーム２４を上端位置まで上昇させ、試料容器ホルダー１を液体窒素２１から吊り上げ、凍結処理後の試料容器５を取り出す。なお、自動昇降機２３によりガイドロッド８を上下移動させる場合に限らず、機械的にアーム２４の上下位置を位置決めするような機構としてもよい。また、手作業によりガイドロッドの上昇及び下降を行ってもよい。 On the other hand, when the lowering button is pressed, the arm 24 is lowered as shown in FIG. Since the sample container holder 1 is freely movable above the stopper 9 of the guide rod 8, the sample container holder 1 floats in the liquid nitrogen 21. In this state, since the sample container 5 set in the sample container holder 1 is immersed in the liquid nitrogen 21, a freezing process is performed.
Thereafter, the arm 24 is raised to the upper end position by pushing the up button, the sample container holder 1 is lifted from the liquid nitrogen 21, and the frozen sample container 5 is taken out. The mechanism is not limited to the case where the guide rod 8 is moved up and down by the automatic elevator 23 but may be a mechanism that mechanically positions the vertical position of the arm 24. Further, the guide rod may be raised and lowered manually.

以上のような凍結処理装置により、凍結処理が施される生体試料は特に制限されるものではなく、ＥＳ細胞、ｉＰＳ細胞、生殖細胞等が挙げられる。また、試料容器の形状は特に制限されるものではなく、チューブ状容器、ストロー状容器、細胞シートを内部に封入可能な袋状容器等、目的に応じて任意のものを用いることができる。また、保持部材の形状も、容器の形状にあわせて適宜変更することができる。ストロー状容器等のように液体窒素中へ浸漬させたとき浮き上がりやすい構造のものを利用する場合は、必要に応じて、ストロー状容器の上端を押さえる部材を配置してもよい。   The biological sample subjected to the freezing process by the freezing apparatus as described above is not particularly limited, and examples thereof include ES cells, iPS cells, germ cells and the like. The shape of the sample container is not particularly limited, and any sample container can be used depending on the purpose, such as a tube-shaped container, a straw-shaped container, a bag-shaped container in which a cell sheet can be enclosed. Further, the shape of the holding member can be appropriately changed according to the shape of the container. When using a structure that easily floats when immersed in liquid nitrogen, such as a straw-shaped container, a member that holds the upper end of the straw-shaped container may be disposed as necessary.

次に、実施例により本発明をより具体的に説明する。尚、本発明はこれらの実施例に限定されるものではない。
（１）装置
図１、４、及び５の概念図に示す試料容器ホルダーを作製した。
（ｉ）試料容器ホルダー
・上側保持部材：発泡塩化ビニル板
直径：７６ｍｍφ、厚さ：３ｍｍ、比重：０．７ｇ／ｃｍ２
生体容器の挿入孔（１４ｍｍφ）が等間隔で８箇所設けられている。
・下側保持部材：下記の第１プレート部材（発泡塩化ビニル板）と第２プレート部材（発泡樹脂板）を使用した。
発泡塩化ビニル板：直径：７６ｍｍφ、厚さ：３ｍｍ、比重：０．７ｇ／ｃｍ２
発泡樹脂板：発泡スチロール製、直径：７６ｍｍφ、厚み：１０ｍｍ、
比重：０．０１５ｇ／ｃｍ２
上側保持部材と、下側保持部材の中央部に、ガイドロッドを貫通させるための１４ｍｍφの貫通孔が設けられている。
・固定支柱：ガラス繊維強化エポキシ樹脂製、本数：４本
サイズ：３ｍｍφ、長さ：４０ｍｍ
（ii）ガイドロッド：ガラス繊維強化樹脂製、
サイズ：１２ｍｍφ、長さ：３００ｍｍ
（iii）デュワー容器
ガラス製、透明デュワー瓶（サーモス（株）製、Ｄ−１０００Ｇ型φ１６０ｍｍ×Ｈ２７６ｍｍ）
（iv）自動昇降装置
モーター駆動方式自動昇降装置（昇降範囲：１００ｍｍ、昇降速度：１００ｍｍ／３ｓｅｃ） Next, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples.
(1) Apparatus A sample container holder shown in the conceptual diagram of FIGS.
(I) Sample container holder / upper holding member: foamed vinyl chloride plate Diameter: 76 mmφ, thickness: 3 mm, specific gravity: 0.7 g / cm 2
Eight insertion holes (14 mmφ) are provided at equal intervals in the living body container.
Lower holding member: The following first plate member (foamed vinyl chloride plate) and second plate member (foamed resin plate) were used.
Foamed vinyl chloride plate: Diameter: 76 mmφ, thickness: 3 mm, specific gravity: 0.7 g / cm 2
Foam resin plate: made of polystyrene foam, diameter: 76 mmφ, thickness: 10 mm,
Specific gravity: 0.015 g / cm 2
A through hole of 14 mmφ for penetrating the guide rod is provided in the central portion of the upper holding member and the lower holding member.
・ Fixed support: Made of glass fiber reinforced epoxy resin, Number: 4 Size: 3mmφ, Length: 40mm
(Ii) Guide rod: Made of glass fiber reinforced resin,
Size: 12mmφ, Length: 300mm
(Iii) Dewar container Glass, transparent dewar (Thermos Co., Ltd., D-1000G type φ160 mm × H276 mm)
(Iv) Automatic lifting device Motor driven automatic lifting device (lifting range: 100mm, lifting speed: 100mm / 3sec)

（２）試料容器内の生体試料の凍結操作
以下の操作により、試料容器内の生体試料を凍結した。
（ｉ）デュワー容器内に液体窒素を貯留した。
（ii）上側保持部材と下側保持部材のそれぞれの貫通孔を貫通させて配置されたガイドロッドの上部先端は、自動昇降装置のアームと取り付けた。
（iii）試料容器（ＦＡＬＣＯＮ社製、内容積：１．８ｍＬ）内に生体試料として細胞を採取した後、速やかに試料容器ホルダーに装着し、自動昇降装置の下降釦を押して、約３０ｍｍ／秒の速さでアームを下降させることにより、デュワー内の液体窒素に試料容器ホルダーを浮遊させた。尚、試料容器の約３分の２は液体窒素に浸漬されていた。３０秒間試料容器ホルダーをデュワー内の液体窒素に浮遊させた後に、自動昇降装置の上昇釦を押し試料容器ホルダーを液体窒素中から引き上げた。
（iv）試料容器ホルダーから試料容器を取外し、液体窒素で冷却された移送容器内に保存した。 (2) Freezing operation of biological sample in sample container The biological sample in the sample container was frozen by the following operation.
(I) Liquid nitrogen was stored in a Dewar container.
(Ii) The upper end of the guide rod disposed through the through holes of the upper holding member and the lower holding member was attached to the arm of the automatic lifting device.
(Iii) After collecting cells as a biological sample in a sample container (product of FALCON, internal volume: 1.8 mL), quickly attach it to the sample container holder, and press the lowering button of the automatic lifting device, about 30 mm / second The sample container holder was suspended in the liquid nitrogen in the dewar by lowering the arm at a speed of. In addition, about two-thirds of the sample container was immersed in liquid nitrogen. After the sample container holder was suspended in the liquid nitrogen in the dewar for 30 seconds, the lift button of the automatic lifting device was pushed to lift the sample container holder out of the liquid nitrogen.
(Iv) The sample container was removed from the sample container holder and stored in a transfer container cooled with liquid nitrogen.

本発明の予備凍結装置は、バイオメディカル基礎研究、再生医療向けのヒト細胞等の生体試料凍結保存分野の研究で生体試料を予備凍結するのに極めて有用である。
また、バイオリソース（生物遺伝資源）の予備凍結処理工程を迅速、安全かつ簡素な操作で行うことが可能である。 The preliminary freezing apparatus of the present invention is extremely useful for preliminary freezing of a biological sample in the field of biomedical basic research and biological sample cryopreservation fields such as human cells for regenerative medicine.
In addition, it is possible to perform a preliminary freezing treatment process of bioresources (biological genetic resources) with a quick, safe and simple operation.

１ 試料容器ホルダー
２ 保持部材
３ 上側保持部材
４ 下側保持部材
５ 試料容器
６ キャップ
７ 固定支柱
８ ガイドロッド
９ ストッパー
１１ 挿入孔
１２ 貫通孔
１３ 貫通孔
１４ 貫通孔
１５ 第１プレート部材
１６ 第２プレート部材
１７ 第１プレート部材
１８ 第２プレート部材
２１ 液体窒素
２２ 真空断熱槽
２３ 自動昇降機
２４ アーム DESCRIPTION OF SYMBOLS 1 Sample container holder 2 Holding member 3 Upper side holding member 4 Lower side holding member 5 Sample container 6 Cap 7 Fixed support | pillar 8 Guide rod 9 Stopper 11 Insertion hole 12 Through-hole 13 Through-hole 14 Through-hole 15 1st plate member 16 2nd plate Member 17 First plate member 18 Second plate member 21 Liquid nitrogen 22 Vacuum heat insulation tank 23 Automatic elevator 24 Arm

Claims (6)

液体窒素液面近傍に浮遊可能な、試料容器ホルダーと、それを保持するガイドロッドとを備えた、試料容器を液体窒素に直接浸漬させて試料容器内の生体試料を凍結するための予備凍結装置であって、
（ｉ）試料容器ホルダーには試料容器を保持する保持部材が設けられており、
（ii）保持部材の一部もしくは全部が液体窒素に浮遊可能な部材から形成され、
（iii）保持部材には、ガイドロッドを貫通させるための貫通孔が設けられており、
（iv）ガイドロッドの下端には試料容器ホルダーの落下防止用ストッパーが設けられていて、
（ｖ）ガイドロッドが保持部材の貫通孔を貫通して配置されることにより、試料容器ホルダーをストッパーより上側で移動可能に支持している
ことを特徴とする、生体試料の予備凍結装置。 A preliminary freezing device for freezing a biological sample in a sample container by directly immersing the sample container in liquid nitrogen, comprising a sample container holder capable of floating near the liquid nitrogen liquid surface and a guide rod for holding the sample container holder Because
(I) The sample container holder is provided with a holding member for holding the sample container,
(Ii) a part or all of the holding member is formed of a member that can float in liquid nitrogen;
(Iii) The holding member is provided with a through hole for allowing the guide rod to pass therethrough,
(Iv) A stopper for preventing the sample container holder from falling is provided at the lower end of the guide rod,
(V) The biological sample preliminary freezing apparatus, wherein the guide rod is disposed through the through hole of the holding member to support the sample container holder so as to be movable above the stopper. 前記試料容器ホルダーが、試料容器を保持するための保持部材として、１以上の試料容器を貫通して保持するための挿入孔を有する上側保持部材、試料容器の底部を支持する下側保持部材、及び上側保持部材と下側保持部材とを連結する１又は２以上の固定支柱を備え、
前記上側保持部材および下側保持部材の相対する位置にはガイドロッドを貫通させるための貫通孔がそれぞれ設けられており、
ガイドロッドが前記貫通孔を貫通することにより、試料容器ホルダーをストッパーより上側で移動可能な状態で支持している
ことを特徴とする、請求項１に記載の生体試料の予備凍結装置。 As the holding member for holding the sample container, the sample container holder has an upper holding member having an insertion hole for penetrating and holding one or more sample containers, a lower holding member for supporting the bottom of the sample container, And one or more fixed struts for connecting the upper holding member and the lower holding member,
Through holes for penetrating the guide rods are respectively provided at the opposing positions of the upper holding member and the lower holding member,
2. The biological sample preliminary freezing apparatus according to claim 1, wherein the sample container holder is supported so as to be movable above the stopper by penetrating the through hole. 上側保持部材と下側保持部材のいずれか一方又は双方の一部もしくは全部が液体窒素に浮遊可能な部材で形成されていることを特徴とする、請求項２に記載の生体試料の予備凍結装置。   The biological sample preliminary freezing apparatus according to claim 2, wherein a part or all of one or both of the upper holding member and the lower holding member is formed of a member capable of floating in liquid nitrogen. . 下側保持部材の下部側に浮力調節手段が配置されていることを特徴とする、請求項２又は３に記載の生体試料の予備凍結装置。   4. The biological sample preliminary freezing apparatus according to claim 2, wherein buoyancy adjusting means is disposed on the lower side of the lower holding member. 前記ガイドロッドがガラスエポキシ樹脂、ポリテトラフルオロエチレン樹脂、もしくはフェノール樹脂、又はこれらの樹脂の繊維強化複合材料であることを特徴とする、請求項１から４のいずれかに記載の生体試料の予備凍結装置。   The biological sample spare according to any one of claims 1 to 4, wherein the guide rod is a glass epoxy resin, a polytetrafluoroethylene resin, a phenol resin, or a fiber-reinforced composite material of these resins. Freezing equipment. 前記ガイドロッドの上端が自動昇降機に接続されていることを特徴とする、請求項１から５のいずれか記載の生体試料の予備凍結装置。
The biological sample preliminary freezing apparatus according to any one of claims 1 to 5, wherein an upper end of the guide rod is connected to an automatic elevator.
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