JP7511344B2 - 単一細胞を封入する方法、封入された細胞およびその使用 - Google Patents
単一細胞を封入する方法、封入された細胞およびその使用 Download PDFInfo
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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- A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M25/16—Particles; Beads; Granular material; Encapsulation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0012—Cell encapsulation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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Description
いくつかの実施形態において、中空ビーズは、ハイドロゲル組成物から調製されたポリマーシェルを有する。本明細書で使用される場合、用語「ハイドロゲル」は、有機ポリマー(天然または合成)が共有結合、イオン結合、または水素結合を介して架橋されて、水分子を捕捉してゲルを形成する三次元開格子構造を作るときに形成される物質を指す。いくつかの実施形態において、ハイドロゲルは、生体適合性ハイドロゲルであってもよい。本明細書で使用される場合、用語「生体適合性ハイドロゲル」は、生細胞に対して毒性を示さず、生存能を維持するための捕捉細胞への酸素および栄養素の十分な拡散を可能にするゲルを形成するポリマーを指す。いくつかの実施形態において、ハイドロゲル材料には、アルギン酸、アクリルアミド、またはポリエチレングリコール(PEG)、PEG-アクリレート、PEG-アミン、PEG-カルボン酸、PEG-ジチオール、PEG-エポキシド、PEG-イソシアネート、PEG-マレイミド、ポリアクリル酸(PAA)、ポリ(メタクリル酸メチル)(PMMA)、ポリスチレン(PS)、ポリスチレンスルホン酸(PSS)、ポリビニルピロリドン(PVPON)、N,N’-ビス(アクリロイル)シスタミン、ポリプロピレンオキシド(PPO)、ポリ(メタクリル酸ヒドロキシエチル)(PHEMA)、ポリ(N-イソプロピルアクリルアミド)(PNIPAAm)、ポリ(乳酸)(PLA)、ポリ(乳酸-グリコール酸共重合体)(PLGA)、ポリカプロラクトン(PCL)、ポリ(ビニルスルホン酸)(PVSA)、ポリ(L-アスパラギン酸)、ポリ(L-グルタミン酸)、ポリリジン、寒天、アガロース、ヘパリン、硫酸化アルギン酸、デキストラン硫酸、ヒアルロナン、ペクチン、カラギーナン、ゼラチン、キトサン、セルロース、コラーゲン、ビスアクリルアミド、ジアクリレート、ジアリルアミン、トリアリルアミン、ジビニルスルホン、ジエチレングリコールジアリルエーテル、エチレングリコールジアクリレート、ポリメチレングリコールジアクリレート、ポリエチレングリコールジアクリレート、トリメチルプロパントリメタクリレート、エトキシ化トリメチロールトリアクリレート、またはエトキシ化ペンタエリスリトールテトラアクリレート、またはその組合せもしくは混合物が挙げられる。いくつかの実施形態において、ハイドロゲルは、アルギン酸、アクリルアミド、またはPEGベースの材料である。いくつかの実施形態において、ハイドロゲルは、アクリル酸-ジチオール、エポキシド-アミン反応化学を用いるPEGベースの材料である。いくつかの実施形態において、ハイドロゲルは、PEG-マレイミド/ジチオール油(dithiol oil)、PEG-エポキシド/アミン油、PEG-エポキシド/PEG-アミン、またはPEG-ジチオール/PEG-アクリレートを含むポリマーシェルを形成する。いくつかの実施形態において、ハイドロゲル材料は、細胞内生体分子を損傷する可能性があるフリーラジカルの生成を避けるために選択される。いくつかの実施形態において、ハイドロゲルポリマーは、60~90%の流体、例えば水、および10~30%のポリマーを含む。特定の実施形態において、ハイドロゲルの水含量は約70~80%である。本明細書で使用される場合、数値を修飾する場合の用語「約」または「およそ」は、数値に生じ得る変動を指す。例えば、変動は、特定の基質または成分の製造の違いにより生じ得る。1つの実施形態において、用語「約」は、挙げられた数値の1%、5%、または最大10%以内を意味する。
本明細書で提供されるいくつかの実施形態は、単一細胞をポリマーシェル内に封入して連続性粒子を形成する方法に関する。いくつかの実施形態において、連続性粒子内に封入される細胞を含有する試料が得られる。いくつかの実施形態において、細胞は封入前に固定剤で連続性粒子内に固定することができる。本明細書で使用される場合、固定剤は、細胞を固定することができる薬剤を一般に指す。例えば、固定細胞は、細胞中のタンパク質複合体、核酸複合体、またはタンパク質-核酸複合体を安定化することができる。適切な固定剤および架橋剤には、アルコールまたはアルデヒドベースの固定剤、ホルムアルデヒド、グルタルアルデヒド、エタノールベースの固定剤、メタノールベースの固定剤、アセトン、酢酸、四酸化オスミウム、重クロム酸カリウム、クロム酸、過マンガン酸カリウム、水銀剤、ピクリン酸塩、ホルマリン、パラホルムアルデヒド、アミン反応性NHS-エステル架橋剤、例えば、ビス[スルホスクシンイミジル]スベレート(BS3)、3,3’-ジチオビス[スルホスクシンイミジルプロピオネート](DTSSP)、エチレングリコールビス[スルホスクシンイミジルスクシネート](スルホ-EGS)、ジスクシンイミジルグルタレート(DSG)、ジチオビス[スクシンイミジルプロピオネート](DSP)、ジスクシンイミジルスベレート(DSS)、エチレングリコールビス[スクシンイミジルスクシネート](EGS)、NHS-エステル/ジアジリン架橋剤、例えば、NHS-ジアジリン、NHS-LC-ジアジリン、NHS-SS-ジアジリン、スルホ-NHS-ジアジリン、スルホ-NHS-LC-ジアジリン、およびスルホ-NHS-SS-ジアジリンが挙げられ得る。いくつかの実施形態において、細胞の固定は、細胞の内部状態を保護し、これにより細胞を連続性粒子内に封入中の細胞の修飾を防ぐ。
本明細書で提供されるいくつかの実施形態は、連続性粒子内に封入された単一細胞で複数の連続同時アッセイを行う方法に関する。いくつかの実施形態において、方法は、本明細書に記載されているように連続性粒子を得ること、および連続性粒子内に封入された単一細胞を試薬と連続的に接触させて複数の連続同時アッセイを行うことを含む。
本明細書で提供されるシステム、方法および組成物のいくつかの実施形態は、アダプターが標的核酸にライゲートされる方法を含む。アダプターは、シーケンシングプライマー結合部位、増幅プライマー結合部位、およびインデックスを含んでもよい。例えば、アダプターは、P5配列、P7配列、またはその補体を含んでもよい。本明細書で使用される場合P5配列は、配列番号1(AATGATACGGCGACCACCGA)によって定義された配列を含み、P7配列は、配列番号2(CAAGCAGAAGACGGCATACGA)によって定義された配列を含む。いくつかの実施形態において、P5またはP7配列は、1~20個、例えば1~15個、または1~10個のヌクレオチド長、例えば2、3、4、5、6、7、8、9、または10個のヌクレオチド長であり得るスペーサーポリヌクレオチドをさらに含んでもよい。いくつかの実施形態において、スペーサーは10個のヌクレオチドを含む。いくつかの実施形態において、スペーサーは、10TスペーサーなどのポリTスペーサーである。スペーサーヌクレオチドは、ポリヌクレオチドの5’末端に含まれてもよく、ポリヌクレオチドの5’末端との結合を介して適切な支持体に結合され得る。結合は、ポリヌクレオチドの5’末端に存在するホスホロチオエートなどの硫黄含有求核剤を通じて達成することができる。いくつかの実施形態において、ポリヌクレオチドは、ポリTスペーサーおよび5’ホスホロチオエート基を含むであろう。故に、いくつかの実施形態において、P5配列は、5’ホスホロチオエート-TTTTTTTTTTAATGATACGGCGACCACCGA-3’(配列番号3)であり、いくつかの実施形態において、P7配列は、5’ホスホロチオエート-TTTTTTTTTTCAAGCAGAAGACGGCATACGA-3’(配列番号4)である。
連続性粒子の調製
以下の例は、単一細胞を封入する連続性粒子をマイクロ流体液滴生成器を用いて調製する実施形態を示す。
連続性粒子で行われる同時アッセイ
以下の例は、実施例1からの連続性粒子で行われる、SCI-seq、ATAC-seq、コンビナトリアルインデックス付け、および単一細胞全ゲノム増幅を含む例示的なアッセイを示す。
連続性粒子における核酸ライブラリー調製
以下の例は、連続性粒子内に封入された細胞から全ゲノムライブラリーを調製するための方法を示す。
Claims (23)
- 単一細胞を封入する中空ビーズであって、中空ビーズは、
PEG-エポキシドおよびアミン、またはPEG-エポキシドおよびPEG-アミンを反応させて得られたポリマーシェルと、
ポリマーシェル内に置かれた単一細胞と
を含み、ポリマーシェルが、単一細胞を保持しながらポリマーシェルを通じて試薬の拡散を可能にする細孔を含む、中空ビーズ。 - ポリマーシェルの内部が、水性環境を含む、請求項1に記載の中空ビーズ。
- ポリマーシェル内に置かれた単一細胞が、ポリマーシェルとの相互作用を受けない、および/またはポリマーシェルと接触していない、請求項1に記載の中空ビーズ。
- 中空ビーズが20μm~200μmの直径を有する、請求項1に記載の中空ビーズ。
- ポリマーシェルが、4アームポリエチレングリコール(PEG)を含む、請求項1に記載の中空ビーズ。
- 単一細胞が哺乳動物細胞である、請求項1に記載の中空ビーズ。
- 試薬が、酵素、化学物質、および50塩基対未満の大きさを有するプライマーを含む、請求項1に記載の中空ビーズ。
- 試薬が、リゾチーム、プロテイナーゼK、ランダムヘキサマー、ポリメラーゼ、Φ29 DNAポリメラーゼ、Taqポリメラーゼ、Bsuポリメラーゼ、トランスポザーゼ、Tn5トランスポザーゼ、プライマー、P5アダプター配列プライマー、P7アダプター配列プライマー、リガーゼ、触媒酵素、デオキシヌクレオチド三リン酸、緩衝液、または二価カチオンを含む、請求項1に記載の中空ビーズ。
- 単一細胞を中空ビーズ内に封入する方法であって、
単一細胞をスペーサー油中でポリマーと混合して混合物(a)を形成すること;
混合物(a)を架橋油と混合して、単一細胞を封入するポリマーシェルを形成することであって、ここでポリマーシェルが、単一細胞を保持しながらポリマーシェルを通じて試薬の拡散を可能にする細孔を含み、ここでポリマーがPEG-エポキシドであり、かつ架橋油が油に溶解されたアミン、または、PEG-アミンである、形成すること
を含む、方法。 - 中空ビーズが20μm~200μmの直径を有する、請求項9に記載の方法。
- ポリマーが、4アームポリエチレングリコール(PEG)を含む、請求項9に記載の方法。
- スペーサー油が、鉱油またはフッ化炭素油を含む、請求項9に記載の方法。
- 単一細胞が哺乳動物細胞である、請求項9に記載の方法。
- 試薬が、酵素、化学物質、および50塩基対未満の大きさを有するプライマーを含む、請求項9に記載の方法。
- 試薬が、リゾチーム、プロテイナーゼK、ランダムヘキサマー、ポリメラーゼ、Φ29 DNAポリメラーゼ、Taqポリメラーゼ、Bsuポリメラーゼ、トランスポザーゼ、Tn5トランスポザーゼ、プライマー、P5アダプター配列プライマー、P7アダプター配列プライマー、リガーゼ、触媒酵素、デオキシヌクレオチド三リン酸、緩衝液、界面活性剤、または二価カチオンを含む、請求項9に記載の方法。
- 混合が、単一細胞、ポリマー、スペーサー油、および架橋油を液滴生成器に投入することを含む、請求項9に記載の方法。
- 液滴生成器がマイクロ流体チップである、請求項16に記載の方法。
- 単一細胞が、固定剤との接触による混合の前に固定される、請求項9に記載の方法。
- 固定剤が、メタノールまたはエタノールなどのアルコール、またはパラホルムアルデヒドなどのアルデヒドを含む、請求項18に記載の方法。
- 中空ビーズ内に封入された単一細胞で複数の連続同時アッセイを行う方法であって、
請求項1から8のいずれか一項に記載の単一細胞を封入する中空ビーズを得ることと、
単一細胞を試薬と連続的に接触させて複数の連続同時アッセイを行うことと
を含む、方法。 - 複数の連続同時アッセイが、溶解、DNA解析、RNA解析、タンパク質解析、タグメンテーション、核酸増幅、核酸シーケンシング、DNAライブラリー調製、シーケンシングを用いたトランスポザーゼ接近可能なクロマチンアッセイ(ATAC-seq)、連続性保存転移(CPT-seq)、単一細胞コンビナトリアルインデックス付けシーケンシング(SCI-seq)、もしくは単一細胞ゲノム増幅、または連続的に行われるそれらの任意の組合せを含む、請求項20に記載の方法。
- 単一細胞を封入する中空ビーズが固体支持体上に播種される、請求項20に記載の方法。
- 固体支持体が、エッチング表面、ウェル、フローセル装置、マイクロ流体流路、ビーズ、またはカラムである、請求項22に記載の方法。
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