JP6492106B2 - 3d組織培養のための細胞の調製方法 - Google Patents
3d組織培養のための細胞の調製方法 Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Description
本発明は、3次元組織培養の分野に関する。
3D(3次元)の組織培養は、生物の細胞が、全3次元で、成長または、その周囲の環境との相互作用が許可される、人工的に作成された環境である。
3Dの培養は、様々な理由で、2D(2次元)培養(「単一層」とも呼ばれる)を超えた改良である。生体組織において、細胞は、複雑な(intricate)、細胞間や細胞−マトリックスの相互作用、および栄養素と細胞の複雑なトランスポート力学(dynamics)を有する、3次元の微小環境に存在する。標準的な2Dまたは単一層の細胞培養は、この環境の不適切な表現(representations)である。
これらのマトリックスは、細胞が、生体組織において移動する場合と同様の方法で、細胞が、3D構造内を移動することを補助する。3D構造は、したがって、細胞遊走、分化、生存と成長のための改良されたモデルである。さらに、2Dでは、細胞は、部分的にのみ極性化できるので、3D組織培養は、細胞の極性化のより正確な描写を提供する。後者は、医薬品の有効性と毒性のスクリーニングに使用される、3D組織培養において、好適な細胞のタイプの1つである、肝細胞(hepatocytes)に、特に重要である。さらに、3Dで育った細胞は、2次元で育った細胞よりも、異なった遺伝子発現を示す(ケルムら(Kelm et al) 2010年、リッドキー(Ridky)ら 2010年、コーディ(Cody et. al.)ら 2008年)。
通常の溶質の拡散とエフェクタータンパク質(effector proteins)(成長因子および酵素のような)への結合は、また、3Dの細胞性マトリックスに依存するので、組織規模溶質濃度勾配(tissue scale solute concentration gradients)を確立するために重要である。
a)適切な表面に、細胞をプレーティングする(plating)工程、
b)必要に応じて、上記表面に接着する上記細胞の能力をチェックする工程、
c)上記表面に接着しなかった上記細胞を除去する(discarding)工程、および
d)接着細胞を切離し(detaching)、そして、3D(三次元)組織培養プロセスにそれらを移動する工程。
・スカホルド−ベース(scaffold-based)の3D組織培養、
・ハイドロゲル−ベースの3D組織培養、
・細胞の自己組織化(cellular self-assembly)3D組織培養、
・3Dバイオプリンティング(3D bioprinting)
・初代細胞(primary cells)、
・幹細胞(stem cells)、および/または、
・不死化細胞(immortalized cells)。
・ヒト細胞、
・カニクイザル(cynomolgus)の細胞、
・ブタ細胞、
・イヌ(canine)細胞、および/または
・ラット細胞。
・スカホルド−ベース(scaffold-based)の3D組織培養、
・ハイドロゲル−ベースの3D組織培養、
・細胞の自己組織化(cellular self-assembly)3D組織培養、および/または、
・3Dバイオプリンティング(3D bioprinting)
そして、当該プロセスにおいて、本発明に係る方法に従って調製された細胞が使用される。
・薬の有効性および/または毒性のスクリーニング、
・調査/機構の毒物学(investigative/mechanistic toxicology)、
・ターゲットの発見/識別(target discovery/identification)、
・医薬品リポジショニング研究(drug repositioning studies)
・薬物動態および薬力学のアッセイ(pharmacokinetics and pharmacodynamics assays)、および/または
・再生医療(regenerative medicine)。
・低温タンク(cryo-tank)から、選んだ細胞瓶(cell-vial)を取り、そして、水浴(37℃)へ移す;2分でタイマー設定する。
・50mlのチューブに洗浄/解凍培地の40mlをピペットする
・チューブに肝細胞を移し、培地1mlで洗う。
・50mlのチューブを、遠心分離機に配置し;50rcf(600rpmに相当)で、5分間、遠心分離を開始する。
・上清を除去する。
・20ml洗浄緩衝液でペレットを洗浄する。
・50mlのチューブに、慎重に、洗浄/解凍培地3mlを置く。
・2D細胞培養培地で、細胞ペレットを再懸濁する。
・トリパン ブルーなどで、肝細胞を数える。
・プレ播種のために(for pre-seeding)、コラーゲン被覆細胞培養皿を使用する。
・6cmの皿に、肝細胞を播く:100000−250000肝細胞/cm2。cm2あたり、0.05−0.5ml
・CO2を含むインキュベーターに、37℃で置く。
・所望により:無血清の2D培養培地で接着(attachment)後、培地の交換をする。
・12−96時間後(12時間以上の場合、毎日培地交換)、肝細胞を収穫する。
・予め温めておいたリン酸緩衝生理食塩水で、3回、非接着の肝細胞を洗浄して除去する。
・50−500ul/cm2のウェルから、肝細胞を除去(dislodge)するために、コラゲナーゼ/アキュターゼ(accutase)混合物のような細胞剥離液(cell detachment solution)を、培養皿に添加する。
・37℃で、5−30分間(ほとんどの細胞が、表面から剥離されるまで、目視観察)、インキュベートする。
・分散した肝細胞を、注意して、洗浄緩衝液で、事前充填された50mlのチューブに移す。
・常温で、50rcf(600rpmに相当)で、5分間、遠心分離する
・上清を吸引、ペレットに、1−50mlの洗浄培地を加え、そして、穏やかに、そのチューブを回転して、ペレットを再度懸濁する。
・常温で、50rcf(600rpmに相当)で、5分間、遠心分離手順を繰り返す。
・ペレットに、再凝集培地1mlを加え、チューブを穏やかに回転して、肝細胞を溶解する。
・肝細胞数を数える。
A.GravityPLUSTM プレート(Insphero AG社、スイス)の調製
・PBS/アンフォテリシン(Amphothericin)の0.75の15mlで、オムニトレイ(Omnitray)を満たし、加湿パッド(humidifier pad)を加える。
・フレーム(frame)を、オムニトレイ(Omnitray)の上に置く。
・前記プレートに蓋をし、プレートをラベルする(label plate)。
B.細胞懸濁液の調製
・ファルコン チューブ(falcon tubes)に、規定された細胞数の細胞懸濁液を調製(すなわち、ml当たり、25’000Heps/25’000NPC)する。
・チューブを回して(pivoting the tube)、細胞懸濁液を均質化する(Homogenize)。
C.播種(Seeding)
・Viaflo電子マルチ チャンネル ピペット (インテグラ バイオサイエンス社) を使用する。
・Viafloパラメーターを設定する:分配:40ul、速度3、繰返し:(Viafloの96ウェルに対して1回)
・貯留槽(reservoir)における細胞懸濁液を空にする(細胞の適切な分配を確保するため、貯留槽に30ml以上を注がない)。
・GravityPLUSプレートから、蓋を除く。
・細胞懸濁液の前吸引(Prior Aspiration of cell suspension)、細胞の均一な分配のために、貯留槽を優しく回転(gently pivot reservoir)する。
・Viafloプログラムを起動する:繰り返し分配する。
・挿入物(inserts)の上に、水平に、Viafloを置く、挿入物の上に、Viafloで穏やかに圧力をかけ、そのトップに液滴がタップすることを避ける。
・プレートに、蓋を戻す。
Pichard et al. (2006) Human hepatocyte culture. Meth Mol Biol 320: 283-293.
Kelm et al., Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol. Bioeng. 2003, 83, 173-180.
Kelm et al 2010, A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. Journal of Biotechnology
Ridky TW et al., Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia. Nature Medicine 2010
Cody NA et al., Influence of monolayer, spheroid, and tumor growth conditions on chromosome 3 gene expression in tumorigenic epithelial ovarian cancer cell lines.
BMC Med Genomics. 2008
Kelm & Fussenegger, Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol. 2004, 22, 195-202.
Yuhas, J et al., Simplified method for production and growth of multicellular tumor spheroids. Cancer Res. 1977, 37, 3639-3643.
Metzger et al, The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. Cytotherapy. 2011 Sep;13(8):1000-12.
Geckil et al, Engineering hydrogels as extracellular matrix mimics. Nanomedicine (Lond). 2010 April ; 5(3): 469-484.
Carletti E et al, Scaffolds for tissue engineering and 3D cell culture. Methods Mol Biol. 2011;695:17-39.
Fey and Wrzesinski, Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line. Toxicol Sci. 2012 Jun;127(2):403-11
Jensen J, Hyllner J, Bjorquist P. Human embryonic stem cell technologies and drug discovery. J Cell Physiol 2009; 219: 513-519.
Sartipy & Bjorquist. Concise Review: Human Pluripotent Stem Cell-Based Models for Cardiac and Hepatic Toxicity Assessment. Stem Cells 2011; 29 (5): 744-748
Claims (9)
- 3D組織培養のための細胞を調製する方法であって、
a)適切な表面に、初代細胞(primary cells)をプレートする(plating)工程、
b)必要に応じて、上記表面に接着するそれらの細胞の能力をチェックする工程、
c)上記表面に接着しなかった細胞を除去する(discarding)工程、
d)接着した細胞を切離し(detaching)、そして、3D(三次元)組織培養プロセスにそれらを移動する工程、
を含む方法であって、ここで、
その方法は、a)乃至d)の工程全体にわたる、生存性試験(viability assay)の適用を包含せず、
その細胞は、プレートする(plating)前、その間、または、後の何れの時にも、増殖(expanded)させず、および、その細胞は、プレートした(plating)後に、継代(passaged)されない、方法。 - その方法において、3D組織培養プロセスが、
・スカホルド−ベース(scaffold−based)の3D組織培養、
・ハイドロゲル−ベースの3D組織培養、
・細胞の自己組織化(cellular self−assembly)3D組織培養、および/または、
・3Dバイオプリンティング(3D bioprinting)、
からなる群から選択される、少なくとも1つである、請求項1に記載された方法。 - その方法において、前記細胞が、プレートする(plating)前に解凍される(thawed)凍結保存された(cryopreserved)、細胞である、請求項1または2に記載された方法。
- その方法において、前記細胞が、冷凍(frozen)されていない細胞である、請求項1〜3のいずれか1項に記載された方法。
- その方法において、前記細胞が、哺乳類細胞である、請求項1〜4のいずれか1項に記載された方法。
- 哺乳類細胞が、
・ヒト細胞、
・カニクイザル(cynomolgus)の細胞、
・ブタ細胞、
・イヌ(canine)細胞、
・ラット細胞、
からなる群から選択される、請求項5に記載された方法。 - その方法において、前記細胞が、肝細胞である、請求項1〜6のいずれか1項に記載された方法。
- 3D組織培養が、回転楕円体(spheroid)の形状を採用している、請求項1〜7のいずれか1項に記載の方法。
- ・薬の有効性および/または毒性のスクリーニング、
・調査/機構の毒物学(investigative/mechanistic toxicology)、
・ターゲットの発見/識別(target discovery/identification)、
・医薬品リポジショニング研究(drug repositioning studies)
・薬物動態および薬力学のアッセイ(pharmacokinetics and pharmacodynamics assays)、および/または
・再生医療(regenerative medicine)、
からなる群から選択される、少なくとも1つの目的のために、3D組織培養が使用される請求項1〜8のいずれか1項に記載の方法。
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