JP2000201672A - Composition for cryopreservation of nucleated cell - Google Patents
Composition for cryopreservation of nucleated cellInfo
- Publication number
- JP2000201672A JP2000201672A JP11040413A JP4041399A JP2000201672A JP 2000201672 A JP2000201672 A JP 2000201672A JP 11040413 A JP11040413 A JP 11040413A JP 4041399 A JP4041399 A JP 4041399A JP 2000201672 A JP2000201672 A JP 2000201672A
- Authority
- JP
- Japan
- Prior art keywords
- cryopreservation
- composition
- nucleated cells
- cell
- blood
- Prior art date
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、骨髄、末梢血、臍帯血
等から分離した有核細胞を凍結保存するための凍結保存
用組成物に関する。The present invention relates to a cryopreservation composition for cryopreserving nucleated cells separated from bone marrow, peripheral blood, cord blood and the like.
【０００２】[0002]
【従来の技術】近年、白血病などの造血器腫瘍、再生不
良性貧血に見られる造血不能、固形癌の化学療法におけ
る副作用である造血障害に対して、末梢血、骨髄、臍帯
血の中の造血幹細胞を移植することが盛んに行われてい
る。造血幹細胞は移植前まで凍結保存されることが多
く、保存スペース、解凍時の破壊赤血球による副作用が
問題となるため、また造血幹細胞提供者（ドナー）と患
者（レシピエント）の赤血球の型が異なるＡＢＯミスマ
ッチの場合の副作用防止のため、通常凍結前に造血幹細
胞を含む有核細胞の分離（赤血球除去）が行われてい
る。一般に、血液から治療に必要な造血幹細胞を含む有
核細胞の分離には、強遠心により形成した有核細胞を豊
富に含むバフィーコート層を分離する遠心法や、比重液
（例えばファルマシア社製Ｆｉｃｏｌｌ）を用いて造血
幹細胞を含む単核球画分を分離する比重遠心法がよく用
いられる。従来、上記方法により分離した造血幹細胞を
含む有核細胞を凍結保存する場合の凍結保存剤として
は、ＤＭＳＯ（ジメチルスルホキシド）、グリセリンに
代表される細胞内凍害保護剤や、ＨＥＳ（ヒドロキシエ
チルスターチ）、ＰＶＰ（ポリビニルピロリドン）に代
表される細胞外凍害保護剤が、単独或いは２種類以上組
み合わされ、細胞培養用培地に浮遊した細胞と混和して
用いられている。最近では、特開平６−４６８４０号公
報や、文献（Ｅｘｐｅｒｉｅｎｔｉａ３６（１９８０）
１１２２〜１１２４）で、簡便な操作で、長期間の凍結
保存が可能な凍結保存剤が報告されているが、高価な細
胞培養用培地を用い、コストがかさむ問題点がある。ま
た、特開平８−５９４８９号公報では、凍結血液用凍害
防止剤が報告されているが、細胞培養用培地を用いない
という記載は一切なく、また大量の凍害防止剤（２０〜
８０重量％）を加える必要があるため、試薬調整に非常
に手間と時間がかかる問題点がある。2. Description of the Related Art In recent years, hematopoiesis in peripheral blood, bone marrow, and umbilical cord blood against hematopoietic disorders such as leukemia, hematopoietic disorders seen in aplastic anemia, and side effects of chemotherapy for solid tumors. Transplantation of stem cells is actively performed. Hematopoietic stem cells are often cryopreserved before transplantation, which causes problems in storage space, side effects of destructed red blood cells upon thawing, and different types of red blood cells between hematopoietic stem cell donors (donors) and patients (recipients) In order to prevent side effects in the case of ABO mismatch, nucleated cells including hematopoietic stem cells are usually separated (red blood cells removed) before freezing. In general, nucleated cells including hematopoietic stem cells necessary for treatment are separated from blood by centrifugation to separate a buffy coat layer rich in nucleated cells formed by strong centrifugation or by a specific gravity solution (for example, Ficoll manufactured by Pharmacia). ) Is often used to separate a mononuclear cell fraction containing hematopoietic stem cells. Conventional cryopreservatives for cryopreserving nucleated cells including hematopoietic stem cells isolated by the above method include intracellular cryoprotectants represented by DMSO (dimethyl sulfoxide) and glycerin, and HES (hydroxyethyl starch). An extracellular cryoprotectant represented by PVP (polyvinylpyrrolidone) is used alone or in combination of two or more, and is used as a mixture with cells suspended in a cell culture medium. Recently, Japanese Unexamined Patent Application Publication No. 6-46840 and literature (Expertientia 36 (1980))
1122 to 1124), a cryopreservative that can be cryopreserved for a long period of time with a simple operation has been reported. However, there is a problem in that an expensive cell culture medium is used and the cost increases. Japanese Patent Application Laid-Open No. 8-59489 reports an antifreezing agent for frozen blood. However, there is no description that a cell culture medium is not used, and a large amount of an antifreezing agent (20 to
(80% by weight), there is a problem that it takes a lot of trouble and time to adjust the reagent.
【０００３】[0003]
【発明が解決しようとする課題】本発明の課題は、骨
髄、末梢血、臍帯血等の血液から分離した有核細胞を、
安価で且つ簡便に凍結保存可能な凍結保存用組成物を提
供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide nucleated cells separated from blood such as bone marrow, peripheral blood, and umbilical cord blood.
It is an object of the present invention to provide a cryopreservation composition which can be cryopreserved at low cost and easily.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上記課題
を解決すべく鋭意検討した結果、特定の濃度のデキスト
ラン、ジメチルスルホキシド、アルブミンの組み合わせ
により、簡便に調整可能で且つ高価な細胞培養用培地を
含まず安価に、有核細胞を長期間凍結保存可能であると
いう、驚くべき効果を見出した。即ち、凍結時の組成
が、５ｗ／ｖ％以上２０ｗ／ｖ％未満のデキストランと
３ｗ／ｖ％以上２０ｗ／ｖ％以下のジメチルスルホキシ
ドと１ｗ／ｖ％以上１０ｗ／ｖ％以下のアルブミンを含
み、残余が生理的溶液であることを特徴とする、有核細
胞の凍結保存用組成物である。本発明におけるデキスト
ランは、凍結時の最終濃度としては、５ｗ／ｖ％以上２
０ｗ／ｖ％未満、好ましくは７ｗ／ｖ％以上１５ｗ／ｖ
％未満、より好ましくは９ｗ／ｖ％以上１２ｗ／ｖ％未
満である。５ｗ／ｖ％未満の場合、凍害保護効果に影響
し、解凍後の細胞の生存率及び造血機能が著しく低下し
てしまう。一方、２０ｗ／ｖ％以上の場合、本発明にお
けるジメチルスルホキシドとアルブミンとの組み合わせ
においては凍害保護効果は飽和しており、更にデキスト
ランを溶解するのに非常に時間を要してしまう。また、
本発明におけるデキストランの濃度は比較的低濃度のた
め、生理食塩液にデキストランを溶解した市販のデキス
トラン（例えばデキストラン４０注（ミドリ十字社製、
デキストラン平均分子量４万、デキストラン濃度１０ｗ
／ｖ％））が簡便に使用でき、また滅菌されているため
好ましい。本発明におけるジメチルスルホキシドは、凍
結時の最終濃度としては、３ｗ／ｖ％以上２０ｗ／ｖ％
以下、好ましくは５ｗ／ｖ％以上１５ｗ／ｖ％以下、よ
り好ましくは８ｗ／ｖ％以上１２ｗ／ｖ％以下である。
３ｗ／ｖ％未満の場合、凍害保護効果に影響し、解凍後
の細胞の生存率及び造血機能が著しく低下してしまう。
一方、２０ｗ／ｖ％より多くしても、本発明におけるデ
キストランとアルブミンとの組み合わせにおいては凍害
保護効果は飽和しており、更にジメチルスルホキシドそ
のものに細胞毒性があるため、凍結前後での暴露による
生存率、造血機能への悪影響が強くなってしまう。ま
た、細胞凍結用（例えばＳＩＧＭＡ社製ＤＭＳＯ）のも
のが、発熱性物質不含で、且つ滅菌されているため好ま
しい。本発明におけるアルブミンは、凍結時の最終濃度
としては、１ｗ／ｖ％以上１０ｗ／ｖ％以下、好ましく
は２ｗ／ｖ％以上８ｗ／ｖ％以下、より好ましくは３ｗ
／ｖ％以上６ｗ／ｖ％以下である。１ｗ／ｖ％未満の場
合、凍害保護効果に影響し、解凍後の細胞の生存率及び
造血機能が著しく低下してしまう。一方、１０ｗ／ｖ％
より多くしても、本発明におけるデキストランとジメチ
ルスルホキシドとの組み合わせにおいては、凍害保護効
果は飽和してしまう。本発明で言う生理的溶液とは、
０．９％の生理的食塩液、或いは市販のリンゲル液、ロ
ック液等、生理的に等張な溶液を言う。Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, it has been found that a simple combination of dextran, dimethylsulfoxide and albumin at a specific concentration can be adjusted easily and inexpensively. A surprising effect was found that nucleated cells can be cryopreserved for a long time at low cost without using a culture medium. That is, the composition at the time of freezing contains 5 w / v% or more and less than 20 w / v% of dextran, 3 w / v% or more and 20 w / v% or less of dimethyl sulfoxide, and 1 w / v% or more and 10 w / v% or less of albumin, A composition for cryopreservation of nucleated cells, the balance being a physiological solution. The dextran in the present invention has a final concentration of 5 w / v%
0 w / v%, preferably 7 w / v% or more and 15 w / v
%, More preferably 9 w / v% or more and less than 12 w / v%. If it is less than 5 w / v%, the effect of cryoprotection will be affected, and the cell survival rate and hematopoietic function after thawing will be significantly reduced. On the other hand, when the content is 20 w / v% or more, in the combination of dimethyl sulfoxide and albumin according to the present invention, the cryoprotection effect is saturated, and it takes much time to further dissolve dextran. Also,
Since the concentration of dextran in the present invention is relatively low, commercially available dextran obtained by dissolving dextran in physiological saline (for example, Dextran 40 Injection (manufactured by Midori Cross,
Dextran average molecular weight 40,000, dextran concentration 10w
/ V%)) is preferable because it can be used easily and is sterilized. Dimethyl sulfoxide in the present invention has a final concentration at the time of freezing of 3 w / v% or more and 20 w / v%.
Or less, preferably 5 w / v% or more and 15 w / v% or less, more preferably 8 w / v% or more and 12 w / v% or less.
When the content is less than 3 w / v%, the effect on cryoprotection is affected, and the cell survival rate and hematopoietic function after thawing are significantly reduced.
On the other hand, even if it is more than 20 w / v%, the combination of dextran and albumin of the present invention saturates the cryoprotective effect, and furthermore, dimethyl sulfoxide itself has cytotoxicity. Adversely affect the rate and hematopoietic function. In addition, those for cell freezing (for example, DMSO manufactured by SIGMA) are preferable because they do not contain pyrogenic substances and are sterilized. The albumin in the present invention has a final concentration of 1 w / v% or more and 10 w / v% or less, preferably 2 w / v% or more and 8 w / v% or less, more preferably 3 w / v% or less, when frozen.
/ V% or more and 6 w / v% or less. If it is less than 1 w / v%, the effect of cryoprotection will be affected, and the cell survival rate and hematopoietic function after thawing will be significantly reduced. On the other hand, 10w / v%
Even with more, the combination of dextran and dimethyl sulfoxide of the present invention saturates the cryoprotection effect. The physiological solution referred to in the present invention is:
A physiologically isotonic solution such as a 0.9% physiological saline solution or a commercially available Ringer's solution or Locke's solution.
【０００５】本発明における有核細胞とは、細胞内に核
を有する細胞のことを言い、例えば白血球、顆粒球、好
中球、好酸球、好塩基球、骨髄球、赤芽球、巨核球、リ
ンパ球、Ｔリンパ球、ヘルパーＴリンパ球、サプレッサ
ーＴリンパ球、細胞障害性Ｔリンパ球、Ｂリンパ球、Ｎ
Ｋ細胞、ＮＫＴ細胞、単球、マクロファージ、樹状細
胞、造血幹／前駆細胞、破骨細胞、骨芽細胞、骨細胞、
繊維芽細胞、軟骨芽細胞、間葉系幹／前駆細胞（ｓｔｒ
ｏｍａｌ ｓｔｅｍ ｃｅｌｌ）等が挙げられる。ま
た、血液からの有核細胞の分離方法としては、強遠心に
より形成した有核細胞を豊富に含むバフィーコート層を
分離する遠心法や、比重液を用いて単核球画分を分離す
る比重遠心法や、ヒドロキシエチルスターチ等を用いた
赤血球連銭形成させる赤血球沈降法や、細胞分離フィル
ターを用いて有核細胞をフィルターに捕捉後、回収液を
通液して剥離回収する方法等が挙げられるが、これらに
限定されるものではない。[0005] The nucleated cell in the present invention refers to a cell having a nucleus in the cell, for example, leukocyte, granulocyte, neutrophil, eosinophil, basophil, myelocyte, erythroblast, megakaryocyte. Sphere, lymphocyte, T lymphocyte, helper T lymphocyte, suppressor T lymphocyte, cytotoxic T lymphocyte, B lymphocyte, N
K cells, NKT cells, monocytes, macrophages, dendritic cells, hematopoietic stem / progenitor cells, osteoclasts, osteoblasts, osteocytes,
Fibroblasts, chondroblasts, mesenchymal stem / progenitor cells (str
ommal stem cell) and the like. Methods for separating nucleated cells from blood include a centrifugation method for separating a buffy coat layer enriched in nucleated cells formed by strong centrifugation and a specific gravity for separating a mononuclear cell fraction using a specific gravity solution. Centrifugal method, erythrocyte sedimentation method using redox cell starch, etc. using hydroxyethyl starch, etc., method of capturing nucleated cells with a filter using a cell separation filter, and then removing and collecting by passing a collecting liquid, etc. However, the present invention is not limited to these.
【０００６】[0006]
【実施例】以下に実施例により本発明をより詳細に説明
するが、本発明はこれらにより限定されるものではな
い。EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
【実施例１】ヒト臍帯全血から比重遠心法にて分離した
造血幹細胞を含む有核細胞の凍結保存。 細胞分離操作 ＣＰＤ加ヒト臍帯全血から、比重遠心法（ＶＡＣＵＴＡ
ＩＮＥＲ ＣＰＴ）ベクトンディッキンソン社製）にて
単核球を分離した。次に生理的食塩液にて、２回洗浄を
行った後、生理的食塩液に再浮遊させた。 凍結保存剤添加 で得られた細胞浮遊液と表１に示す組成の凍結保存剤
を１：３の割合で氷冷しながら混和した。 ※１；デキストラン４０注（ミドリ十字社製） 平均分子量４万のデキストランを１０％含む生理食塩液 本発明の実施例におけるデキストランは全く上記のもの
を用いた。 凍結保存／解凍 の細胞浮遊液を凍結チューブに充填し、液体窒素中で
１ケ月間凍結保存した。また、解凍は３７℃の温浴中で
急速解凍した。 白血球カウント 自動血球計数装置（コールター社製ヘマトロジーアナラ
イザー）を用い、凍結解凍前後の白血球をカウントし
た。白血球回収率は以下の式で算出した。 白血球回収率（％）＝１００×（凍結解凍後白血球数／
凍結前白血球数） この場合の白血球回収率は８８％であった。 凍結解凍後白血球の生存率 トリパンブルー染色法にて評価した。この場合の生存率
は、８５％であった。 ｉｎ ｖｉｔｒｏ造血機能評価 造血機能評価用のメチルセルロース培地（Ｓｔｅｍ Ｃ
ｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製 Ｍｅｔｈｏｃ
ｕｌｔ ＧＦ Ｈ４４３４Ｖ）に、凍結解凍前後の白血
球浮遊液から白血球を２．５×１０ｅ４／ｍＬとなるよ
うに添加し、これをよく混和して、φ３５ｍｍプラスチ
ックディッシュに１ｍＬ分注した。造血機能を持つ細胞
は上記培地中で培養することによりコロニー（細胞集
団）を形成する。その後、３７℃ＣＯ２インキュベータ
中で１４日間培養後、コロニー数をカウントし、造血機
能は、以下の式で示される凍結解凍前後での総コロニー
の回収率で評価した。 総コロニーの回収率（％）＝白血球回収率×（凍結解凍
後の１ディッシュあたりのコロニー数／凍結前の１ディ
ッシュあたりのコロニー数） この場合の総コロニーの回収率は８３％であった。Example 1 Cryopreservation of nucleated cells including hematopoietic stem cells separated from human umbilical cord whole blood by specific gravity centrifugation. Cell separation operation From CPD-added human umbilical cord whole blood, specific gravity centrifugation (VACUTA)
(INER CPT) manufactured by Becton Dickinson). Next, after washing twice with physiological saline, the cells were resuspended in physiological saline. The cell suspension obtained by adding the cryopreservative was mixed with the cryopreservative having the composition shown in Table 1 at a ratio of 1: 3 while cooling with ice. * 1: Dextran 40 injection (manufactured by Midori Cross) A physiological saline solution containing 10% of dextran having an average molecular weight of 40,000. The above-mentioned dextran was completely used in the examples of the present invention. The cryopreserved / thawed cell suspension was filled into a cryotube and cryopreserved in liquid nitrogen for one month. Thawing was carried out rapidly in a 37 ° C. warm bath. Leukocyte count Leukocytes before and after freeze-thaw were counted using an automatic blood cell counter (hematology analyzer manufactured by Coulter). The leukocyte recovery was calculated by the following equation. Leukocyte recovery rate (%) = 100 × (freezing and thawing leukocyte count /
(Leukocyte count before freezing) In this case, the leukocyte recovery rate was 88%. Survival rate of leukocytes after freeze-thaw was evaluated by trypan blue staining. The survival rate in this case was 85%. In vitro hematopoietic function evaluation Methylcellulose medium (Stem C) for hematopoietic function evaluation
Methoc manufactured by cell Technologies
ulf GF H4434V), leukocytes were added from the leukocyte suspension before and after the freeze-thaw at 2.5 × 10e4 / mL, mixed well, and 1 mL was dispensed into a φ35 mm plastic dish. Cells having a hematopoietic function form colonies (cell populations) by culturing in the above medium. Thereafter, after culturing in a CO 2 incubator at 37 ° C. for 14 days, the number of colonies was counted, and the hematopoietic function was evaluated by the recovery rate of the total colonies before and after freeze-thawing represented by the following formula. Total colony recovery rate (%) = leukocyte recovery rate x (number of colonies per dish after freezing / thawing / number of colonies per dish before freezing) In this case, the recovery rate of total colonies was 83%.
【０００７】[0007]
【実施例２】ヒト臍帯全血からバフィーコート層を回収
することにより得た造血幹細胞を含む有核細胞の凍結保
存。 細胞分離操作 ＣＰＤ加ヒト臍帯全血１００ｍＬ（ＣＰＤ２８ｍＬ）が
充填された血液バッグを、４４００ｇ×１０分の条件で
遠心し、上方の血漿と下方の濃厚赤血球層の中間層の有
核細胞を多く含むバフィーコート層を回収した。 凍結保存剤添加 で得られたバフィーコート層と表２に示す組成の凍結
保存剤を１：３の割合で氷冷しながら混和した。 凍結保存／解凍 実施例１と同じ。 白血球カウント 実施例１と同じ。この場合の白血球回収率は９０％であ
った。 凍結解凍後白血球の生存率 実施例１と同じ。この場合の生存率は、８７％であっ
た。 ｉｎ ｖｉｔｒｏ造血機能評価 実施例１と同じ。この場合の総コロニーの回収率は８５
％であった。 ※２；バフィーコート中のアルブミンの濃度を５ｗ／ｖ
％とし、２５ｗ／ｖ％ヒトアルブミン試薬との合計で示
した（以下の比較例でも同様に計算）。Example 2 Cryopreservation of nucleated cells including hematopoietic stem cells obtained by recovering a buffy coat layer from human umbilical cord whole blood. Cell Separation Procedure A blood bag filled with 100 mL of CPD-added human umbilical cord whole blood (28 mL of CPD) is centrifuged at 4400 g × 10 minutes, and contains a large amount of nucleated cells in the middle layer between the upper plasma and the lower dense red blood cell layer. The buffy coat layer was collected. The buffy coat layer obtained by adding the cryopreservative and the cryopreservative having the composition shown in Table 2 were mixed at a ratio of 1: 3 while cooling with ice. Cryopreservation / thawing Same as in Example 1. Leukocyte count Same as in Example 1. The leukocyte recovery in this case was 90%. Survival rate of leukocytes after freeze-thawing Same as in Example 1. The survival rate in this case was 87%. In vitro evaluation of hematopoietic function Same as Example 1. In this case, the recovery rate of the total colony is 85.
%Met. * 2: The concentration of albumin in buffy coat was 5 w / v.
%, And the total was shown with 25 w / v% human albumin reagent (calculated in the same manner in the following comparative examples).
【０００８】[0008]
【比較例１】実施例２同様、ヒト臍帯全血からバフィー
コート層を回収することにより得た造血幹細胞を含む有
核細胞の凍結保存であるが、凍結保存剤は表３のものを
用いた。 細胞分離操作 実施例１と同じ。 凍結保存剤添加 で得られたバフィーコート層と表３に示す組成の凍結
保存剤を１：３の割合で氷冷しながら混和した。 凍結保存／解凍 実施例１と同じ。 白血球カウント 実施例１と同じ。この場合の白血球回収率は８９％であ
った。 凍結解凍後白血球の生存率 実施例１と同じ。この場合の生存率は、５０％であっ
た。 ｉｎ ｖｉｔｒｏ造血機能評価 実施例１と同じ。この場合の総コロニーの回収率は４０
％であった。 Comparative Example 1 As in Example 2, nucleated cells including hematopoietic stem cells obtained by collecting a buffy coat layer from human umbilical cord whole blood were cryopreserved. . Cell separation operation Same as in Example 1. The buffy coat layer obtained by adding the cryopreservative was mixed with the cryopreservative having the composition shown in Table 3 at a ratio of 1: 3 while cooling with ice. Cryopreservation / thawing Same as in Example 1. Leukocyte count Same as in Example 1. The leukocyte recovery in this case was 89%. Survival rate of leukocytes after freeze-thawing Same as in Example 1. The survival rate in this case was 50%. In vitro evaluation of hematopoietic function Same as Example 1. In this case, the recovery rate of the total colony is 40.
%Met.
【０００９】[0009]
【比較例２】実施例２同様、ヒト臍帯全血からバフィー
コート層を回収することにより得た造血幹細胞を含む有
核細胞の凍結保存であるが、凍結保存剤は表４のものを
用いた。 細胞分離操作 実施例１と同じ。 凍結保存剤添加 で得られたバフィーコート層と表４に示す組成の凍結
保存剤を１：３の割合で氷冷しながら混和した。 凍結保存／解凍 実施例１と同じ。 白血球カウント 実施例１と同じ。この場合の白血球回収率は９０％であ
った。 凍結解凍後白血球の生存率 実施例１と同じ。この場合の生存率は、６０％であっ
た。 ｉｎ ｖｉｔｒｏ造血機能評価 実施例１と同じ。この場合の総コロニーの回収率は５０
％であった。 Comparative Example 2 As in Example 2, nucleated cells including hematopoietic stem cells obtained by recovering a buffy coat layer from human umbilical cord whole blood were cryopreserved. . Cell separation operation Same as in Example 1. The buffy coat layer obtained by adding the cryopreservative and the cryopreservative having the composition shown in Table 4 were mixed at a ratio of 1: 3 while cooling with ice. Cryopreservation / thawing Same as in Example 1. Leukocyte count Same as in Example 1. The leukocyte recovery in this case was 90%. Survival rate of leukocytes after freeze-thawing Same as in Example 1. The survival rate in this case was 60%. In vitro evaluation of hematopoietic function Same as Example 1. The total colony recovery rate in this case is 50
%Met.
【００１０】[0010]
【比較例３】実施例１同様、ヒト臍帯全血から比重遠心
法にて分離して得た造血幹細胞を含む有核細胞の凍結保
存であるが、凍結保存剤は表５のものを用いた。 細胞分離操作 実施例１と同じ。 凍結保存剤添加 で得られた細胞浮遊液と表５に示す組成の凍結保存剤
を１：３の割合で氷冷しながら混和した。 凍結保存／解凍 実施例１と同じ。 白血球カウント 実施例１と同じ。この場合の白血球回収率は８８％であ
った。 凍結解凍後白血球の生存率 実施例１と同じ。この場合の生存率は、５５％であっ
た。 ｉｎ ｖｉｔｒｏ造血機能評価 実施例１と同じ。この場合の総コロニーの回収率は４３
％であった。 表６に結果のまとめを示す。 表６のように、至適濃度のデキストラン、ジメチルスル
ホキシド、アルブミンを添加することにより、凍結解凍
後に高いｉｎ ｖｉｔｒｏ造血機能を維持することがで
きた。Comparative Example 3 As in Example 1, nucleated cells containing hematopoietic stem cells obtained by separation from human umbilical cord whole blood by specific gravity centrifugation were cryopreserved. . Cell separation operation Same as in Example 1. The cell suspension obtained by adding the cryopreservative was mixed with the cryopreservative having the composition shown in Table 5 at a ratio of 1: 3 while cooling with ice. Cryopreservation / thawing Same as in Example 1. Leukocyte count Same as in Example 1. The leukocyte recovery in this case was 88%. Survival rate of leukocytes after freeze-thawing Same as in Example 1. The survival rate in this case was 55%. In vitro evaluation of hematopoietic function Same as Example 1. The total colony recovery rate in this case was 43
%Met. Table 6 summarizes the results. As shown in Table 6, by adding dextran, dimethyl sulfoxide and albumin at optimal concentrations, a high in vitro hematopoietic function could be maintained after freezing and thawing.
【００１１】[0011]
【発明の効果】本発明の凍結保存用組成物は、安価で且
つ簡便な方法で試薬調整でき、しかも有核細胞を長期間
凍結保存できる。EFFECT OF THE INVENTION The composition for cryopreservation of the present invention can be prepared by a low-cost and simple method, and can preserve nucleated cells for a long period of time.
Claims (2)
／ｖ％未満のデキストランと３ｗ／ｖ％以上２０ｗ／ｖ
％以下のジメチルスルホキシドと１ｗ／ｖ％以上１０ｗ
／ｖ％以下のアルブミンを含み、残余が生理的溶液であ
ることを特徴とする、有核細胞の凍結保存用組成物。1. The composition at the time of freezing is 5 w / v% or more and 20 w
Dextran of less than 3% w / v and 20% w / v
% Or less of dimethyl sulfoxide and 1 w / v% or more and 10 w
A composition for cryopreservation of nucleated cells, wherein the composition comprises albumin of at most / v% and the balance is a physiological solution.
あることを特徴とする、請求項１記載の有核細胞の凍結
保存用組成物。2. The composition for cryopreservation of nucleated cells according to claim 1, wherein the nucleated cells are nucleated cells including hematopoietic stem cells.
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| JP11040413A JP2000201672A (en) | 1999-01-11 | 1999-01-11 | Composition for cryopreservation of nucleated cell |
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| JP11040413A JP2000201672A (en) | 1999-01-11 | 1999-01-11 | Composition for cryopreservation of nucleated cell |
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ID=12579989
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| JP11040413A Withdrawn JP2000201672A (en) | 1999-01-11 | 1999-01-11 | Composition for cryopreservation of nucleated cell |
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| Country | Link |
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