JP6581758B2 - 幹細胞及びそのデータを獲得する方法 - Google Patents
幹細胞及びそのデータを獲得する方法 Download PDFInfo
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Description
(外)
ヒト前駆幹細胞濃縮カクテル(RosetteSep
(外)
Human Progenitor Enrichment Cocktail)である場合、試験管内には、2ミリリットル(mL)の全血検体を入れた。引き続き、全血検体を室温(例えば、27℃前後)にて、所定の時間(例えば、20分前後)を培養する。培養完了後、第1溶液を用いて全血検体を希釈し、ゆっくりと両者を混合させる。混合完了後には、希釈試料が得られる。係る第1溶液は、例えば2%の牛胎児血清(fetal bovine serum, FBS)/燐酸緩衝生理食塩水(phosphate buffered saline, PBS)である。引き続き、試薬(例えば、Ficoll−Paque
(外)
)を入れた試験管に希釈試料をゆっくりと入れる。入れる過程では、希釈試料と試薬とが相互に混合してしまうことを避けて、試料が試薬の上面に位置させるようにする。引き続き、希釈試料と試薬とを含有する混合物を室温(例えば、27℃前後)にて、適切な回転速度(例えば、毎分3,000回転)で所定の時間(例えば、20分間)を遠心し、遠心完了後は、4つの顕著な成層構造が形成される。これらの4つの顕著な成層構造は、上から下に向けて、血漿層と、多種の細胞を含む細胞層、試薬層、赤血球層から構成されている。引き続き、血漿層と、多種細胞を含む細胞層と、試薬層との上半分とをもう一つの試験管に入れて、適切な回転速度(例えば、毎分3,000回転)で所定の時間(例えば、20分間)を遠心し、遠心完了後には、遠心試料が得られる。引き続き、遠心試料の上澄み液(supernatant)を取り除き、遠心試料内の複数種細胞を第2溶液(例えば、2%のFBS/PBS)に再び懸濁させる。さらに、適切量の緩衝液(例えば、3X赤血球分解液(red−blood−cell (RBC)lysis buffer)を第2溶液に加え、緩衝液を含まれた第2溶液を室温で所定の時間(例えば、10分間)培養した後に、第3溶液(例えば、2%のFBS/PBS)に用いて、第2溶液(このときは、緩衝液が含まれている)内の複数種細胞を2回洗い流す。洗い流し完了後、複数種の細胞を第4溶液(例えば、1%または2%ウシ血清アルブミン(bovine serum albumin, BSA)を含む燐酸緩衝生理食塩水)に再び懸濁させて、複数種の細胞と第4溶液の混合物を獲得する。本発明において、0.1グラムのウシ血清アルブミンを10ミリリットルの燐酸緩衝生理食塩水緩衝液に溶かせることによって、1%のウシ血清アルブミンを含む燐酸緩衝生理食塩水緩衝液を得ることができる。0.2グラムのウシ血清アルブミンを10ミリリットルの燐酸緩衝生理食塩水緩衝液に溶かせることによって、2%のウシ血清アルブミンを含む燐酸緩衝生理食塩水緩衝液を得ることができる。引き続き、適切な抗体を含む試薬を複数種の細胞と第4溶液の混合物に加えて、試験試料が形成される。試験試料は、以降の解析工程(T0)に使用される。
(外)
(登録商標)溶液であっても良い。血液試料を入れた採血管は、例えば図13のステップ103で獲得することができる。血液試料は、個体(前述の個体(S))の末梢血を採取し、採取した血液試料を前述採血管に入れる。血液試料にステップ1を実行する前に、血液試料は例えば、温度が1〜30℃(例えば、1〜10℃、10〜20℃または20〜30℃)の環境に貯蔵することができる。血液試料を獲得してから、ステップ1の血液試料に対する測量方法MTを行う時間の間隔は、30分、60分、12時間、24時間、48時間、72時間または1週間、例えば、10〜30分、30〜60分、0.5〜2時間、2〜12時間、12〜24時間、24〜48時間または48〜72時間とする。血液試料の体積は、例えば5ミリリットルまたは10ミリリットルより大きいか又は等しい。血液試料と6%のヒドロキシエチル澱粉を含む赤血球凝集剤の体積比は、例えば5:1であっても良い。6%ヒドロキシエチル澱粉は一種の溶液であり、燐酸緩衝生理食塩水(PBS)に6%のヒドロキシエチル澱粉を含有しており、つまり、毎100ミリリットル(mL)の燐酸緩衝生理食塩水(PBS)に6グラムのヒドロキシエチル澱粉を含有している。
を含める種の特定の細胞数に関わる変化量を得ることができる。
11 フィルタ
12a 試験管
12b 試験管
12c 試験管
13 遠心機
14a 試験試料
14b 試験試料
14c 試験試料
15a 解析機器
15b 解析機器
16 赤血球分解液
19 貯蔵器
20a 試験管
20b 試験管
20c 試験管
21a 試験試料
21b 試験試料
21c 試験試料
22 赤血球分解液
1〜20 ステップ
21〜43 ステップ
49〜59 ステップ
61〜70 ステップ
77 垂直線
81 垂直破線
82 水平破線
100a 1ミリメートルの正方形
100b 1ミリメートルの正方形
100c 1ミリメートルの正方形
100d 1ミリメートルの正方形
101〜104 ステップ
Claims (7)
- 幹細胞数を獲得する方法であって、以下のステップ:
被験者から獲得された末梢血試料を試験試料に加工するステップであって、前記加工するステップは、前記末梢血試料を最上層および最下層に分離するステップと、前記最上層から前記試験試料を獲得するステップとを含むステップと、
前記試験試料から第1の部分と第2の部分とを取り出すステップと、
第1のデータを獲得するように、前記第1の部分において粒子の第1のグループをカウントするステップであって、前記カウントするステップは、1.0マイクロメーター以上のサイズを有する細胞の数を獲得するために血球計算器を使用することを含み、前記第1のデータは、前記試験試料において1.0マイクロメーター以上のサイズを有する細胞の単位体積当たりの数である試験試料細胞数を含むステップと、
第2のデータを獲得するように、前記第2の部分における粒子の第2のグループにおける細胞をカウントするステップであって、前記カウントするステップは、前記第2の部分において幹細胞の1つ以上のタイプの各々の数、1.0マイクロメーター以上のサイズを有する細胞の数(細胞数C1)、及び前方散乱係数対側方散乱係数のグラフにおける高い細胞核/細胞質比を有する領域における細胞の数(細胞数C2)を獲得するフローサイトメーターを使用するステップを含み、前記幹細胞の1つ以上のタイプはLgr5(+)幹細胞を含み、前記第2のデータは前記細胞数C1における前記幹細胞の1つ以上のタイプの各々のパーセンテージを含み、前記第2のデータは、
(i)前記細胞数C1における前記細胞数C2のパーセンテージを獲得するステップと、
(ii)前記細胞数C2における前記幹細胞の1つ以上のタイプの各々のパーセンテージを獲得するステップと(パーセンテージSP)、
(iii)C1中のC2のパーセンテージにSP及び100%を掛けるステップと、
を含むプロセスにより獲得されるステップと、
前記第1のデータと前記第2のデータとに基づいて、第3のデータを獲得するステップであって、前記第3のデータは、前記末梢血試料における前記幹細胞の1つ以上のタイプの各々の単位体積当たりの数であり、前記第3のデータは、
(i)C1における前記幹細胞の1つ以上のタイプの各々のパーセンテージに試験試料の細胞数を掛けることにより、前記試験試料における前記幹細胞の1つ以上のタイプの各々の単位体積当たりの数を獲得するステップと、
(ii)体積変化を獲得するため、前記試験試料の体積を前記末梢血試料の体積で割ること、及び前記体積変化に前記試験試料における前記幹細胞の1つ以上のタイプの各々の単位体積当たりの数を掛けることにより、前記末梢血試料における前記幹細胞の1つ以上のタイプの各々の単位体積当たりの数を獲得するステップと、
を含むプロセスにより獲得されるステップと、
を含む、幹細胞数を獲得する方法。 - 前記細胞数C1は、白血球、赤血球、血小板、及び顆粒細胞のカウントを含む、請求項1に記載の方法。
- 前記幹細胞の1つ以上のタイプは、CD24(+)、CD33(+)、CD349(+)、SSEA1(+)、SSEA4(+)、CD9(+)、CD133(+)、またはCD66e(+)幹細胞をさらに含む、請求項1に記載の方法。
- 前記末梢血試料が、前記被験者が行為を行った後に前記被験者から獲得される、請求項1に記載の方法。
- 前記行為は生薬またはフコイダンを含有する組成物を服用することである、請求項4に記載の方法。
- 請求項1に記載の方法であって、前記加工するステップは、
第1の中間試料を獲得するため、EDTA試験管中で、前記末梢血と赤血球凝集剤とを混合するステップと、
前記第1の中間試料を、最上層と最下層とに分離するまでインキュベートするステップと、
前記最上層を収集するステップと、
第1のペレットを獲得するため、前記最上層を遠心分離するステップと、
第2の中間試料を獲得するため、第1の塩溶液に前記第1のペレットを再懸濁させるステップであって、前記第1の塩溶液はカルシウムおよびマグネシウムを含まない、ステップと、
第2のペレットを獲得するため、前記第2の中間試料を遠心分離するステップと、
第3の中間試料を獲得するため、第2の塩溶液に前記第2のペレットを再懸濁させるステップであって、前記第1の塩溶液はカルシウムおよびマグネシウムを含まない、ステップと、
第3のペレットを獲得するため、前記第3の中間試料を遠心分離するステップと、および
前記試験試料を獲得するため、培養液に前記第3のペレットを再懸濁させるステップと、
を含む方法。 - 前記フローサイトメーターを用いて前記幹細胞の1つ以上のタイプを同定するため、前記第2の部分を1つ以上の細胞表面マーカーで標識するステップをさらに含む、請求項6に記載の方法。
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TWI614340B (zh) | 2011-09-28 | 2018-02-11 | 幹細胞生物科技股份有限公司 | 體幹細胞及其製備方法 |
TWI687519B (zh) | 2012-12-06 | 2020-03-11 | 美商幹細胞生物科技股份有限公司 | Lgr5+體幹細胞 |
EP2746770A1 (en) | 2012-12-21 | 2014-06-25 | Stembios Technologies, Inc. | Method for evaluating effect of action on subject based on stem celldynamics |
US20140377760A1 (en) * | 2013-06-24 | 2014-12-25 | StemBios Technologies, Inc. | Method for increasing number of stem cells in human or animal bodies |
WO2016081553A1 (en) * | 2014-11-19 | 2016-05-26 | StemBios Technologies, Inc. | Somatic stem cells for treating bone defects |
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