RU2021118349A - Способы инкапсулирования одиночных клеток, инкапсулированные клетки и способы их применения - Google Patents
Способы инкапсулирования одиночных клеток, инкапсулированные клетки и способы их применения Download PDFInfo
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- 238000000034 method Methods 0.000 title claims 13
- 239000008187 granular material Substances 0.000 claims 12
- 239000011324 bead Substances 0.000 claims 11
- 210000004027 cell Anatomy 0.000 claims 11
- 239000007787 solid Substances 0.000 claims 11
- 239000003921 oil Substances 0.000 claims 8
- 229920000642 polymer Polymers 0.000 claims 7
- 238000004458 analytical method Methods 0.000 claims 4
- 239000004971 Cross linker Substances 0.000 claims 3
- 238000003556 assay Methods 0.000 claims 3
- 239000003153 chemical reaction reagent Substances 0.000 claims 3
- 238000009792 diffusion process Methods 0.000 claims 3
- 238000012163 sequencing technique Methods 0.000 claims 3
- PVVTWNMXEHROIA-UHFFFAOYSA-N 2-(3-hydroxypropyl)-1h-quinazolin-4-one Chemical compound C1=CC=C2NC(CCCO)=NC(=O)C2=C1 PVVTWNMXEHROIA-UHFFFAOYSA-N 0.000 claims 2
- 239000004593 Epoxy Substances 0.000 claims 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims 2
- JMXMXKRNIYCNRV-UHFFFAOYSA-N bis(hydroxymethyl)phosphanylmethanol Chemical compound OCP(CO)CO JMXMXKRNIYCNRV-UHFFFAOYSA-N 0.000 claims 2
- 239000003638 chemical reducing agent Substances 0.000 claims 2
- 238000004132 cross linking Methods 0.000 claims 2
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical group SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 claims 2
- 150000004662 dithiols Chemical class 0.000 claims 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 2
- 102000039446 nucleic acids Human genes 0.000 claims 2
- 108020004707 nucleic acids Proteins 0.000 claims 2
- 150000007523 nucleic acids Chemical class 0.000 claims 2
- 239000011148 porous material Substances 0.000 claims 2
- 230000002441 reversible effect Effects 0.000 claims 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims 2
- ZQXIMYREBUZLPM-UHFFFAOYSA-N 1-aminoethanethiol Chemical compound CC(N)S ZQXIMYREBUZLPM-UHFFFAOYSA-N 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims 1
- 108010077544 Chromatin Proteins 0.000 claims 1
- 108010024636 Glutathione Proteins 0.000 claims 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 claims 1
- 102000008579 Transposases Human genes 0.000 claims 1
- 108010020764 Transposases Proteins 0.000 claims 1
- 150000001412 amines Chemical class 0.000 claims 1
- 230000003321 amplification Effects 0.000 claims 1
- 210000003483 chromatin Anatomy 0.000 claims 1
- 230000009089 cytolysis Effects 0.000 claims 1
- 238000000354 decomposition reaction Methods 0.000 claims 1
- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 claims 1
- 230000005684 electric field Effects 0.000 claims 1
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 claims 1
- 238000013412 genome amplification Methods 0.000 claims 1
- 229960003180 glutathione Drugs 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000002480 mineral oil Substances 0.000 claims 1
- 235000010446 mineral oil Nutrition 0.000 claims 1
- 238000012986 modification Methods 0.000 claims 1
- 230000004048 modification Effects 0.000 claims 1
- DJVKJGIZQFBFGS-UHFFFAOYSA-N n-[2-[2-(prop-2-enoylamino)ethyldisulfanyl]ethyl]prop-2-enamide Chemical group C=CC(=O)NCCSSCCNC(=O)C=C DJVKJGIZQFBFGS-UHFFFAOYSA-N 0.000 claims 1
- 238000003199 nucleic acid amplification method Methods 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 229940071127 thioglycolate Drugs 0.000 claims 1
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 claims 1
- 230000017105 transposition Effects 0.000 claims 1
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- C12N5/0012—Cell encapsulation
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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
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Claims (28)
1. Твердая подложка, включающая множество гранул, где каждая гранула содержит:
полимерную оболочку; и
одиночную клетку, расположенную внутри полимерной оболочки,
где полимерная оболочка содержит поры, которые обеспечивают диффузию реагента через полимерную оболочку с одновременным удержанием одиночной клетки.
2. Твердая подложка по п. 1, где твердая подложка представляет собой устройство с проточными ячейками, планшет с лунками, микропанель, микрофлюидный канал, предметное стекло или структурированная поверхность.
3. Твердая подложка по п. 1, где твердая подложка представляет собой устройство с проточными ячейками, включающее вкладыш с микролунками или микростолбиками в устройстве для распределения множества гранул для поддержания смежности для пространственного индексирования в устройстве с проточными ячейками.
4. Твердая подложка по п. 1, где твердая подложка представляет собой планшет с лунками, включающий множество лунок, где каждая из множеств гранул загружена в каждую лунку из множества лунок.
5. Твердая подложка по п. 1, где планшет с лунками включает от приблизительно 12 до 960 лунок.
6. Твердая подложка по п. 1, где каждая гранула имеет диаметр приблизительно от 20 мкм до приблизительно 200 мкм.
7. Твердая подложка по п. 1, где полимерная оболочка содержит ПЭГ-малеинимид/дитиоловое масло, ПЭГ-эпоксид/аминовое масло, ПЭГ-эпоксид/ПЭГ-амин, или ПЭГ-дитиол/ПЭГ-акрилат.
8. Полая гранула по п. 1, где полимерная оболочка содержит обратимый кросслинкер.
9. Полая гранула по п. 1, где обратимый кросслинкер представляет собой N,N’-бис(акрилoил)цистамин.
10. Способ анализа отдельных клеток в популяции клеток, включающий:
контактирование популяции клеток с полимером в разделительном масле для образования смеси (a);
перемешивание смеси (a) с маслом для перекрестного сшивания для образования множества гранул, каждая гранула инкапсулирует одиночную клетку, где каждая гранула содержит поры, которые обеспечивают диффузию реагента через гранулу с одновременным удержанием одиночной клетки;
контактирование множества гранул с реагентом для выполнения множества последовательных совместных анализов популяции клеток;
разделение гранул на твердой подложке; и
модификация разделенных гранул, чтобы обеспечить диффузию инкапсулированного материала из разделенных гранул.
11. Способ по п.10, где модификация разделенных гранул включает повышение температуры гранул.
12. Способ по п.11, в котором температуру повышают более чем 50°C.
13. Способ по п.10, где модификация разделенных гранул включает разрушение крослинкера гранул.
14. Способ по п.13, где разложение включает приведение гранул в контакт с восстановителем.
15. Способ по п.14, где восстановитель представляет собой дитиоэритритол (DTE), дитиотрейтол (DTT), 2-меркаптоэтанол, аминоэтантиол, глутатион, тиогликолат, 2,3-димеркаптопропанол, трис(2-карбоксиэтил)фосфин (TCEP), трис(гидроксиметил)фосфин (THP), или P-[трис(гидроксиметил)фосфин] пропионовую кислоту (THPP).
16. Способ по п.10, где модификация разделенных гранул включает воздействие на гранулы электрического поля.
17. Способ по п. 10, где множественные последовательные совместные анализы включают лизис, анализ ДНК, анализ РНК, анализ белков, тагментацию, амплификацию нуклеиновых кислот, секвенирование нуклеиновых кислот, получение библиотеки ДНК, анализ для оценки хроматина, доступного для транспозазы, с использованием секвенирования (ATAC-seq), транспозицию, сохраняющую смежность (CPT-seq), комбинаторное индексированное секвенирование одиночной клетки (SCI-seq), или амплификацию генома одиночной клетки, или любое их сочетание, проводимое последовательно.
18. Способ по п. 10, где каждая гранула имеет диаметр приблизительно от 20 мкм до приблизительно 200 мкм.
19. Способ по п. 10, где разделительное масло включает минеральное масло или фторуглеродное масло.
20. Способ по п.10, в котором смешивание включает помещение одиночной клетки, полимера, разделительного масла и масла для перекрестного сшивания в генератор капель, где необязательно генератор капель необязательно представляет собой микрофлюидный чип.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201862660452P | 2018-04-20 | 2018-04-20 | |
| US62/660,452 | 2018-04-20 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| CN111051525A (zh) | 2020-04-21 |
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| US20190352591A1 (en) | 2019-11-21 |
| JP7511344B2 (ja) | 2024-07-05 |
| JP7542570B2 (ja) | 2024-08-30 |
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| MX2019014802A (es) | 2021-03-02 |
| KR20240052875A (ko) | 2024-04-23 |
| US12480090B2 (en) | 2025-11-25 |
| RU2750567C2 (ru) | 2021-06-29 |
| KR20210153770A (ko) | 2021-12-17 |
| KR102481869B1 (ko) | 2022-12-27 |
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