Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine
冷冻保存的人类全肠组织免疫细胞分离、类器官构建及共培养建立方案
(*contributed equally to this work) 发布: 2025年01月05日第15卷第1期 DOI: 10.21769/BioProtoc.5157 浏览次数: 2345
评审: Andrea GramaticaPhilipp WörsdörferAnonymous reviewer(s)
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Q&A
参见作者原研究论文
The authors used this protocol in:
Mar 2024
Abstract
The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures.
Key features
• Dissection and tissue analysis of whole human intestines.
• Cryopreservation for biobank generation.
• Optimized protocols for the isolation of epithelial and immune cells.
• Autologous co-culture of organoids and lamina propria–derived immune cells.
Keywords: Whole gut collection (全肠收集)
Tissue processing (组织处理)
Biobank (生物样本库)
Cryopreservation (冷冻保存)
Immune cell isolation (免疫细胞分离)
Intestinal organoid (肠道类器官)
Autologous co-culture (自体共培养)
Lamina propria (固有层)
Intestinal immune organoids (肠道免疫类器官)
Graphical overview
Human intestine processing to cryopreserve biopsies, isolate epithelial and immune cells, and generate organoids and autologous co-culture. Created with BioRender.com.
Background
The intestine is the longest organ of the human body and possesses the largest surface area, being also constantly exposed to commensal and pathogenic bacteria. Thus, maintaining barrier integrity and accurate immune surveillance is crucial to preserve its function and homeostasis. A dysfunction in the epithelial barrier or in the immune cell composition of the intestine can lead to severe and chronic diseases, from cancer to autoimmune diseases. Therefore, it is essential to understand the interaction between the epithelial barrier and the immune cells as well as to identify region-specific immune cell subpopulations.
Access to intestinal tissue is limited, and most research relies on small biopsies (<5 cm) or intestinal resections (<40 cm long) [1–3]. However, to fully understand the complexity and regional variations, studying the intestine in its entirety is necessary. Additionally, this allows the analysis of the jejunum and ileum, which are particularly difficult to address with biopsies or resections.
Processing such a large organ requires a stringent protocol that focuses on the cryopreservation and time-independent analysis of the samples. Moreover, the focus of most cryopreservation methods is on the recovery of a specific cell population [4,5], but they do not address the isolation of cells from different intestinal regions or simultaneous epithelial and immune cell isolation [3,6].
Our protocol closes this gap by describing a cryopreservation method that supports the preservation of both immune and epithelial cells of various intestinal regions while additionally being applicable to cell isolations from fresh tissue. It serves as an essential and versatile guideline for the preservation and cell isolation of the whole human intestine while providing additional options for tissue analysis.
We give a detailed description of an optimized and refined protocol that describes how to process the whole intestine, generate a biobank, and perform cell isolation from fresh and cryopreserved tissue. In particular, techniques for the generation of intestinal organoids and the isolation of epithelium and immune cells with enrichment of specific cell fractions depending on the enzyme digestion carried out are presented. Finally, we provide a protocol to establish an autologous immune cell-organoid co-culture, which can be further modified to study one specific intestinal region or immune cell subtype.
Materials and reagents
Biological materials
1. Human whole intestinal tissues (Novobiosis, I3PT biobank, DTI Foundation)
2. R-Spondin 1 cell line, stably transfected HEK293T cells (kindly provided by Hugo de Jonge)
Reagents
General buffers and media
1. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.2)
2. Potassium di-hydrogen phosphate (KH2PO4) (Carl Roth, catalog number: P018.2)
3. Di-sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) (Carl Roth, catalog number: T877.1)
4. Heat-inactivated FBS (Capricorn Scientific, catalog number: FBS-HI-11A)
5. PBS-/- (Capricorn Scientific, catalog number: PBS-1A)
6. HEPES (Capricorn Scientific, catalog number: HEP-B)
7. 0.5 M EDTA pH 8.0 (Fisher Scientific, catalog number: 46-034-CI)
8. Sucrose for molecular biology, ≥99.5% (GC) (Sigma-Aldrich, catalog number: S0389-500G)
9. RPMI 1640 medium, 500 mL (Thermo Fisher Scientific, catalog number: 21875034)
10. Custodiol (Dr. Franz Köhler Chemie)
11. DTT (1,4-Dithiothreitol) (Carl Roth, catalog number: 6908.3)
12. Ethanol Rotipuran ≥99.8% p.a. (Carl Roth, catalog number: 9065.05)
13. Methanol (Carl Roth, catalog number: 8388.6)
Buffers and media for cryopreservation
1. Isopropanol 99.8% (neoLab Migge, catalog number: 2-3701)
2. CryoSure-DMSO USP grade (WAK-Chemie Medical, catalog number: WAK-DMSO-10)
3. Ethylene glycol anhydrous, 99.8% (Sigma-Aldrich, catalog number: 324558-100ml)
4. D-(+)-trehalose dihydrate (Sigma-Aldrich, catalog number: T0167-25G)
5. RNA later stabilization solution (Qiagen, Fisher Scientific, catalog number: AM7021)
6. CryoStor® CS10 (STEMCELL Technologies, catalog number: 07930)
Chemicals for histology
1. FSC 22 frozen section media, blue, 9 × 118 mL (Leica, catalog number: 3801481)
2. Paraformaldehyde solution 4% in PBS (Santa Cruz Biotechnology, catalog number: sc-281692)
3. Paraplast (Paraffin) (Carl Roth, catalog number: X880.2)
4. Xylene (Sigma-Aldrich, catalog number: 534056)
5. Tissue freezing medium (Leica, catalog number: 14020108926)
Buffers and media for organoid generation
1. Matrigel growth factor reduced (GFR) basement membrane matrix, phenol red-free, LDEV-free (Corning, Fisher Scientific, catalog number: 356231)
2. StemProTM AccutaseTM cell dissociation reagent (Thermo Fisher, Gibco, catalog number: A1110501)
3. Advanced DMEM/F12 medium (Fisher Scientific, Gibco, catalog number: 12634010)
4. GlutaMAX (Thermo Fisher Scientific, catalog number: 35050038)
5. Primocin (Invivogen, catalog number: ant-pm-1)
6. B-27 supplement (Thermo Fisher Scientific, catalog number: 17504044)
7. WNT surrogate-Fc fusion protein (ImmunoPrecise, catalog number: N001)
8. R-Spondin 1-conditioned medium (produced by R-Spondin 1 cell line)
9. n-Acetyl-cysteine (Sigma-Aldrich, catalog number: A7250)
10. Nicotinamide (Sigma-Aldrich, catalog number: N0636-500G)
11. Recombinant murine EGF (PeproTech, catalog number: 315-09)
12. Murine noggin (Peprotech, catalog number: 250-38)
13. Recombinant human IGF-1 (PeproTech, catalog number: 100-11)
14. Recombinant human FGF-2 (PeproTech, catalog number: 100-18C)
15. Human Leu[15]-Gastrin I (Sigma-Aldrich, catalog number: G9145)
16. A83-01 (Sigma-Aldrich, catalog number: SML0788)
17. SB202190 (Tocris, catalog number: 1264)
18. Y-27632 (Hölzel Diagnostika, catalog number: HY-10583)
Buffers and media for cell isolation
1. HBSS without Ca2+/Mg2+ (Thermo Fisher Scientific, catalog number: 14175129)
2. Multi Tissue Dissociation kit (Miltenyi BioTec, catalog number: 130-110-201)
3. Collagenase type IV (Fisher Scientific, catalog number: 17104019)
4. DNase I (Sigma-Aldrich, Chemie, catalog number: 10104159001)
5. LiberaseTM TL research grade (Sigma-Aldrich, catalog number: 5401020001)
Flow cytometric analysis
1. CD45 BV510 (BD Biosciences, catalog number: 563204)
2. EPCAM (CD326) PE (BioLegend, catalog number: 324206)
3. CD3 BUV496 (BD Biosciences, catalog number: 612940)
4. CD19 BV750 (BioLegend, catalog number: 302262)
5. HLADR BUV395 (BD Biosciences, catalog number: 564040)
6. CD11c BB515 (BD Biosciences, catalog number: 564490)
7. CD14 BUV737 (BD Biosciences, catalog number: 612763)
8. CD56 APC/Cy7 (BioLegend, catalog number: 318332)
9. CD103 APC (BioLegend, catalog number: 350216)
10. 7AAD (BioLegend, catalog number: 420404)
Supplies for co-culture
1. Percoll (Thermo Fisher Scientific, catalog number: 17-0891-01)
2. Glutaraldehyde 50% (Sigma-Aldrich, Chemie, catalog number: G7776-10ML)
3. Formaldehyde solution 37% (Carl Roth, catalog number: CP10.1)
Solutions
1. 10× phosphate-buffered saline (PBS) (see Recipes)
2. Cryopreservation buffer (see Recipes)
3. Complete Roswell Park Memorial Institute medium (cRPMI) (see Recipes)
4. Mucus removal solution (see Recipes)
5. 30% ethanol (see Recipes)
6. 50% ethanol (see Recipes)
7. 70% ethanol (see Recipes)
8. 80% ethanol (see Recipes)
9. 5% sucrose (see Recipes)
10. 15% sucrose (see Recipes)
11. 30% sucrose (see Recipes)
12. Dissociation solution (see Recipes)
13. Base medium (see Recipes)
14. Growth medium (see Recipes)
15. Expansion medium (see Recipes)
16. 0.75 M sucrose (see Recipes)
17. 0.375 M sucrose (see Recipes)
18. 0.125 M sucrose (see Recipes)
19. 10% FBS/PBS (see Recipes)
20. Epithelial cell removal media (see Recipes)
21. Enzymatic mixture A (see Recipes)
22. Enzymatic mixture B (see Recipes)
23. Enzymatic mixture C (see Recipes)
24. 80% Percoll (see Recipes)
25. 40% Percoll (see Recipes)
26. Co-culture fixative (see Recipes)
Recipes
All buffers can be stored at room temperature unless otherwise specified. All sucrose buffers can be stored at 4 °C and used within 4 weeks.
1. 10× phosphate-buffered saline (PBS)
| Reagent | Final concentration | Amount |
|---|---|---|
| NaCl | 1.37 M | 80.063 g |
| KH2PO4 | 0.018 M | 2.45 g |
| Na2HPO4·2H2O | 0.103 M | 18.333 g |
| H2O | n/a | 900 mL |
| Total | 1 L |
Adjust the pH of 1× PBS to 7.4.
2. Cryopreservation buffer
Adapted from Hughes et al. [4]. Store at 4 °C and use within 72 h.
| Reagent | Final concentration | Amount |
|---|---|---|
| D-(+)-Trehalose dihydrate | 50 mM | 3.78 g |
| CryoSure-DMSO USP grade | 6% | 12 mL |
| Ethylene glycol anhydrous, 99.8% | 5% | 10 mL |
| Heat-inactivated FBS | 178 mL | |
| Total | 200 mL |
3. Complete Roswell Park Memorial Institute medium (cRPMI)
Store at 4 °C and use within 4 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| RPMI | n/a | 450 mL |
| FBS | 5% | 50 mL |
| Total | 500 mL |
4. Mucus removal solution
Store at 4 °C and use within 12 h.
| Reagent | Final concentration | Amount |
|---|---|---|
| RPMI | n/a | 450 mL |
| FBS | 5% | 50 mL |
| DTT (100%) | 4 mM | 0.3 g |
| Total | 500 mL |
5. 30% ethanol
| Reagent | Final concentration | Amount |
|---|---|---|
| Ethanol (absolute) | 30% | 300 mL |
| H2O | n/a | 700 mL |
| Total | 1,000 mL |
6. 50% ethanol
| Reagent | Final concentration | Amount |
|---|---|---|
| Ethanol (absolute) | 50% | 500 mL |
| H2O | n/a | 500 mL |
| Total | 1,000 mL |
7. 70% ethanol
| Reagent | Final concentration | Amount |
|---|---|---|
| Ethanol (absolute) | 70% | 700 mL |
| H2O | n/a | 300 mL |
| Total | 1,000 mL |
8. 80% ethanol
| Reagent | Final concentration | Amount |
|---|---|---|
| Ethanol (absolute) | 80% | 800 mL |
| H2O | n/a | 200 mL |
| Total | 1,000 mL |
9. 5% sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 5% | 5 g |
| PBS | n/a | 100 mL |
| Total | 100 mL |
10. 15% sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 15% | 15 g |
| PBS | n/a | 100 mL |
| Total | 100 mL |
11. 30% sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 30% | 30 g |
| PBS | n/a | 100 mL |
| Total | 100 mL |
12. Dissociation solution
| Reagent | Final concentration | Amount |
|---|---|---|
| PBS-/- | n/a | 9.8 mL |
| EDTA (0.5 M) | 10 mM | 200 μL |
| Total | 10 mL |
13. Base medium
Store at 4 °C and use within 4 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| Advanced DMEM/F12 | n/a | 489 mL |
| HEPES | 10 mM | 5 mL |
| GlutaMAX (100×) | 1× | 5 mL |
| Primocin | 100 μg/mL | 1 mL |
| Total | 500 mL |
14. Growth medium
Store at 4 °C and use within 2 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| Advanced DMEM/F12 | n/a | 6.5379 mL |
| R-Spondin 1-conditioned medium | 20% | 2 mL |
| B-27 supplement | 1× | 200 μL |
| HEPES | 10 mM | 100 μL |
| GlutaMAX (100×) | 1× | 100 μL |
| Nicotinamide | 10 mM | 100 μL |
| Murine noggin | 100 ng/mL | 50 μL |
| N-acetyl-cysteine | 1.25 mM | 25 μL |
| Primocin | 100 μg/mL | 20 μL |
| Surrogate WNT | 0.5 nM | 10 μL |
| EGF | 50 ng/mL | 10 μL |
| SB202190 | 10 μM | 3.3 μL |
| A83-01 | 500 nM | 1 μL |
| Human Leu[15]-Gastrin I | 10 nM | 1 μL |
| Y-27632* | 10 μM | 1 μL |
| Total | 10 mL |
*First 2–3 days after isolation or expansion.
15. Expansion medium
Store at 4 °C and use within 2 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| Advanced DMEM/F12 | n/a | 7.472 mL |
| R-Spondin 1-conditioned medium | 20% | 2 mL |
| B-27 supplement | 1× | 200 μL |
| HEPES | 10 mM | 100 μL |
| GlutaMAX (100×) | 1× | 100 μL |
| Murine noggin | 100 ng/mL | 50 μL |
| N-acetyl-cysteine | 1 mM | 20 μL |
| Primocin | 100 μg/mL | 20 μL |
| Surrogate WNT | 0.5 nM | 10 μL |
| EGF | 50 ng/mL | 10 μL |
| Recombinant human IGF-1 | 100 ng/mL | 10 μL |
| Recombinant human FGF-2 | 50 ng/mL | 5 μL |
| A83-01 | 500 nM | 1 μL |
| Human Leu[15]-Gastrin I | 10 nM | 1 μL |
| Y-27632* | 10 μM | 1 μL |
| Total | 10 mL |
*First 2–3 days after isolation or expansion.
16. 0.75 M sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 0.75 M | 5.13 g |
| PBS | 90% | 18 mL |
| FBS | 10% | 2 mL |
| Total | 20 mL |
17. 0.375 M sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 0.375 M | 2.6 g |
| PBS | 90% | 18 mL |
| FBS | 10% | 2 mL |
| Total | 20 mL |
18. 0.125 M sucrose
| Reagent | Final concentration | Amount |
|---|---|---|
| Sucrose | 0.125 M | 0.9 g |
| PBS | 90% | 18 mL |
| FBS | 10% | 2 mL |
| Total | 20mL |
19. 10% FBS/PBS
Store at 4 °C and use within 72 h.
| Reagent | Final concentration | Amount |
|---|---|---|
| PBS | 90% | 36 mL |
| FBS | 10% | 4 mL |
| Total | 40 mL |
20. Epithelial cell removal media
Store at 4 °C and use within 72 h.
| Reagent | Final concentration | Amount |
|---|---|---|
| HBSS (-/-) | n/a | 39.5 mL |
| HEPES (1 M) | 100 mM | 5 mL |
| FBS | 10% | 5 mL |
| EDTA (0.5 M) | 5 mM | 500 μL |
| Total | 50 mL |
21. Enzymatic mixture A
Prepare directly before use.
| Reagent | Final concentration | Amount |
|---|---|---|
| cRPMI | n/a | 4.725 mL |
| Liberase (2.5 mg/mL) | 100 μg/mL | 200 μL |
| DNase (10 mg/mL) | 150 μg/mL | 75 μL |
| Total | 5 mL |
22. Enzymatic mixture B
Prepare directly before use.
| Reagent | Final concentration | Amount |
|---|---|---|
| cRPMI | n/a | 4.89 mL |
| Collagenase IV (0.327 g/mL) | 2 mg/mL | 31 μL |
| DNase (10 mg/mL) | 150 μg/mL | 75 μL |
| Total | 5 mL |
23. Enzymatic mixture C (Multi Tissue Dissociation kit 1: MTKD1)
Prepare directly before use.
| Reagent | Final concentration | Amount |
|---|---|---|
| cRPMI | n/a | 4.675 mL |
| Enzyme A (MTDK1) | n/a | 25 μL |
| Enzyme R (MTDK1) | n/a | 100 μL |
| Enzyme D (MTDK1) | n/a | 200 μL |
| Total | 5 mL |
24. 80% Percoll
Store at 4 °C and use within 4 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| Percoll (100%) | 80% | 72 mL |
| 10× PBS | n/a | 8 mL |
| PBS-/- | n/a | 20 mL |
| Total | 100 mL |
25. 40% Percoll
Store at 4 °C and use within 4 weeks.
| Reagent | Final concentration | Amount |
|---|---|---|
| Percoll (100%) | 80% | 54 mL |
| 10× PBS | n/a | 6 mL |
| PBS-/- | n/a | 40 mL |
| Total | 100 mL |
26. Co-culture fixative
Prepare on the day of use.
| Reagent | Final concentration | Amount |
|---|---|---|
| Paraformaldehyde solution 37% in PBS | 3.7% | 1 mL |
| Glutaraldehyde 50% | 0.1% | 20 μL |
| PBS-/- | 8.98 mL | |
| Total | 10 mL |
Laboratory supplies
General
1. 15 mL tubes (neoLab Migge, catalog number: 352096)
2. 50 mL tubes (neoLab Migge, catalog number: 352070)
3. 5 mL serological pipette (Falcon, neoLab Migge, catalog number: 357543)
4. 10 mL serological pipette (Falcon, neoLab Migge, catalog number: 357551)
5. 25 mL serological pipette (Falcon, neoLab Migge, catalog number: 357525)
6. Cell strainer 100 μm (Greiner Bio-One, catalog number: 542000)
7. Cell strainer 70 μm (Greiner Bio-One, catalog number: 542070)
8. FACS tubes, Falcon 5 mL round-bottom polystyrene test tube, with cell strainer snap cap (Fisher Scientific, catalog number: 352235)
9. SafeSeal tubes 1.5 mL (Sarstedt, catalog number: 72.706)
Supplies for lab preparation
1. Autoclaving bags, large size (Greiner Bio-One, catalog number: 649201)
2. Labeling tape (neoLab Migge, catalog number: 2-6101)
3. Sterile drape Absorba-II, 200 × 320 cm (Integra, catalog number: 2206-3114)
4. Plastic tray (neoLab Migge, catalog number: 2-3853)
5. Forceps, small (Vubu Medical Instruments, catalog number: VUBU-02-45911 and VUBU-02-8911)
6. Forceps, big (Vubu Medical Instruments, catalog number: VUBU-02-21921 and VUBU-02-67920)
7. Intestine scissors (Vubu Medical Instruments, catalog number: VUBU-03-30521)
8. Scissors, curved (Vubu Medical Instruments, catalog number: VUBU-03-16114)
9. Scissor small (Vubu Medical Instruments, catalog number: VUBU-03-44908)
10. Petri dish 9 cm (Carl Roth, catalog number: N221.2)
11. Cooling element, Va-Q-accu 27L +05G (Va-Q-tec, catalog number: AK000295)
12. Ice bucket with lid (neoLab, catalog number: 2-6002)
13. Dry ice nuggets (CLEANGAS)
14. Measuring tape (Amazon, BLAMT, Stanley, catalog number: 0-34-295)
15. Safety pin 4.5 cm (Amazon, TK Gruppe Timo Kringler, catalog number: B082ZQZTDQ)
16. Wide metal dish 29 × 23 × 4 cm (Rewe, catalog number: 32420)
17. Bench cover, Foliodrape protect cover cloth 45 × 75 cm (Medpex, catalog number: 798179)
18. Labcoat (neoLab Migge, catalog number: 4-1482)
19. Protective gowns (Carl Roth, catalog number: CAL9.1)
20. Sleeves Medi-Inn (Carl Roth, catalog number: 1PP4.1)
21. Plastic box 15.7 × 11.8 × 10.0 cm, 1 L (Amazon, Rotho, catalog number: 6116705070)
Supplies for tissue collection
1. Freezing container, 18 × 1–2 mL (neoLab Migge, catalog number: 2-3701)
2. Barcoded cryovials, 2 mL (Greiner Bio-One, catalog number: 122280-2DG)
3. Scanner (CSL-Computer, catalog number: 722304674722 via Amazon)
4. Black cryo rack (Greiner Bio-One, catalog number: 802576)
5. White cryo rack (Greiner Bio-One, catalog number: 802202)
6. Beaker 800 mL (neoLab Migge, catalog number: E1052)
7. Sealing film parafilm M, 100 mm, 38 m (Carl Roth, catalog number: H666.1)
8. S-Monovette Serum-Gel 7.5 mL (Sarstedt AG & Co. KG, catalog number: 01.1602)
9. Cotton swaps, sterile (P+W Medizintechnik Platz & Co Handels GmbH, catalog number: 10724)
Supplies for histology
1. Histology cassettes (neoLab Migge, catalog number: 70012)
2. TT Cryomold intermediate, 15 × 15 × 15 mm (Science Service, catalog number: SA62534-15)
3. BD Microlance 3 Nr. 20, 27 G needle (MSG Medizinische Geräte, Handel und Service, catalog number: BD302200)
Supplies for organoid generation
1. 24-well plate (Greiner Bio-One, catalog number: 662160)
2. 200 μL pipette tips without filter (Sarstedt, catalog number: 70.760.202)
Supplies for immune cell isolation
1. Cell strainer 40 μm (Greiner Bio-One, neoLab Migge, catalog number: 352340)
2. 35 mL GosselinTM X500 FIOLE DROSO 25X95MM VRAC PS PS (Fisher Scientific, catalog number: 11773303)
3. Magnetic stirring bar, PTFE, cylindrical (BRAND, Merck Chemicals, catalog number: Z328723-10EA)
4. Neubauer counting chamber (neoLab Migge, catalog number: 191844172)
5. Duran crystallizing beaker (Merck Chemicals GmbH, catalog number: Z231754)
6. C-tubes for gentleMACS (Miltenyi BioTec, catalog number: 130-093-237)
7. Round-bottom 96-well plates (neoLab Migge, catalog number: C-8207)
Equipment
1. Pipet boy acu 2 (Integra BioscienceTM, catalog number: 10060262)
2. Cell culture incubator (Heracell, catalog number: 51032720)
3. Herasafe 2030i biosafety hood (Thermo Fisher Scientific, catalog number: 51032324)
4. Forced convection laboratory incubator (ScanCell, catalog number: 37)
5. 5810 R centrifuge (Eppendorf, catalog number: 5811000420)
6. Fluorescence microscope (Nikon, model: Eclipse Ti2)
7. Microscope stage incubator (Tokai Hit)
8. Rocker shaker 2D series (Carl Roth GmbH+Co. KG, catalog number: XT47.1)
9. IKA 4011000 Roller shaker (IKA, catalog number: EW-04304-05)
10. CryoStar NX50 (Thermo Fisher Scientific, EprediaTM, catalog number: 13435949)
11. HM 340E Microtome (Thermo Fischer Scientific, catalog number: 26370735)
12. gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec B.V., catalog number: 130-096-427)
13. Spectral Analyzer (SONY, model: ID7000TM)
14. Electrothermal Paraffin section flotation bath (Cole-Parmer, catalog number: MH8517)
15. Orbital shaker (Sunlab, catalog number: SU1030)
16. Scale (Sartorious, catalog number: CP32025-OCE)
17. Vortexer (Neolab, catalog number: D6012)
18. -80 °C freezer
19. -20 °C freezer
20. 4 °C fridge
21. N2 tank
22. Paraffin processing station: Leica, ASP 300S Tissue Processor
23. Casting station: Leica, HistoCore Arcadia H Embedding Center
24. Magnetic stirrer with heater (IKA, model: RCT5 digital)
Software and datasets
1. FlowJo v10.8.1
2. GraphPad Prism v.10.0.0
3. Fiji (ImageJ)
4. Microsoft Excel
Procedure
文章信息
稿件历史记录
提交日期: Jun 20, 2024
接收日期: Oct 28, 2024
在线发布日期: Nov 20, 2024
出版日期: Jan 5, 2025
版权信息
© 2025 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Gamero-Estevez, E., Hensel, I. V., Steinhauer, M., Müllertz, O., Savochkina, E., Sektioglu, I. M., Stoll, B., Derakhshani, S., Devriese, S., Kim, K. and Resnik-Docampo, M. (2025). Protocol for Immune Cell Isolation, Organoid Generation, and Co-culture Establishment from Cryopreserved Whole Human Intestine. Bio-protocol 15(1): e5157. DOI: 10.21769/BioProtoc.5157.
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分类
细胞生物学 > 细胞分离和培养 > 共培养
细胞生物学 > 细胞分离和培养 > 器官培养
干细胞 > 类器官培养
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