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The present study describes methodologies for culturing, freezing, thawing, processing, staining, labeling, and examining entire spheroids and organoids under various microscopes, while they remain intact in a hydrogel within a multipurpose device.
Organoids and spheroids, three-dimensional growing structures in cell culture labs, are becoming increasingly recognized as superior models compared to two-dimensional culture models, since they mimic the human body better and have advantages over animal studies. However, these studies commonly face problems with reproducibility and consistency. During the long experimental processes - with transfers of organoids and spheroids between different cell culture vessels, pipetting, and centrifuging - these susceptible and fragile 3D growing structures are often damaged or lost. Ultimately, the results are significantly affected, since the 3D structures cannot maintain the same characteristics and quality. The methods described here minimize these stressful steps and ensure a safe and consistent environment for organoids and spheroids throughout the processing sequence while they are still in a hydrogel in a multipurpose device. The researchers can grow, freeze, thaw, process, stain, label, and then examine the structure of organoids or spheroids under various high-tech instruments, from confocal to electron microscopes, using a single multipurpose device. This technology improves the studies' reproducibility, reliability, and validity, while maintaining a stable and protective environment for the 3D growing structures during processing. In addition, eliminating stressful steps minimizes handling errors, reduces time taken, and decreases the risk of contamination.
The future of cell research and therapy lies within 3D cell cultures1,2,3. Organoid and spheroid models close the gap between in vitro experiments and animal models by creating better models that mimic human body development, physiology, and diseases4,5,6,7,8,9. However, the reproducibility and repeatability of these models remain challenging. Furthermore, handling, harve....
1. Culturing organoids and spheroids
The present article represents a multipurpose device (MD) and complementing methodologies for culturing, freezing, thawing, histological staining, immunohistochemical staining, immunofluorescence labeling, coating, and processing of entire organoids or spheroids while still in a hydrogel in a single uniquely designed environment. The current study was designed to prepare HepG2 liver cancer spheroids in 35 hydrogel drops in 35 MDs. Experiments were conducted in triplicate to ensure accuracy. Additionally, lung organoids i.......
The MD, complementing formulations and protocols described here, facilitates rapid and spontaneous 3D growth of organoids and spheroids in a more controlled environment and continues the experiment in the same conditions. The specimen stays in the same environment during the entire process, and nearly 100% of the 3D growing architectures remain intact in the container. This improves the homogeneity during the sequential experiments and allows for an extended culture period. In addition, the number of steps during organoi.......
We are grateful to Dale Mertes from the University of Chicago for the preparation of diagrams, to Dr. Mehmet Serif Aydin for his technical support at Istanbul Medipol University Research Institute for Health Sciences and Technologies, and to Dr. Rana Kazemi from Maltepe University for editing the manuscript.
....Name | Company | Catalog Number | Comments |
Absolute Ethanol (EtOH) | Merck | 8187602500 | Dilute in dH2O to make 30%, 50%, 70%, 80%, 90% and 96% solution and store at RT |
Acetone | Merck | 8222512500 | Store at RT |
Alexa fluor wheat germ agglutinin and Hoechst in Hank's balanced salt solution (HBSS)  | Invitrogen | I34406 | Image-IT LIVE Plasma Membrane and Nuclear Labeling Kit, Store at -20 °C |
Alpha-1-Fetoprotein (AFP) Concentrated and Prediluted Polyclonal Antibody | Biocare Medical | CP 028 A | Store at +4 °C |
Anti-albumin antibody | Abcam | EPR20195 | Store at +4 °C, Dilution: 1:50 |
Anti-beta galactosidase antibody, Chicken polyclonal | Abcam | 134435 | Store at +4 °C, Dilution: 1:25 |
Anti-cytokeratin 5 | Abcam | 53121 | Store at +4 °C, Dilution: 1:100 |
Arginase-1 Concentrated and Prediluted Rabbit Monoclonal Antibody | Biocare Medical | ACI 3058 A, B | Store at +4 °C, Dilution: 1:50 |
Calcium chloride (CaCl2) | Sigma | C1016-500G | Dissolve in Karnovsky's fixative to make 2 mM CaCl2; store at RT |
Cell Counting Kit 8 (WST-8 / CCK8) | Abcam | ab228554 | |
Centrifuge tubes, 15 mLÂ | Nest | 601051 | |
Centrifuge tubes, 50 mLÂ | Nest | 602052 | |
Class II Microbiological Safety Cabinet Bio II Advance Plus | Telstar | EN12469 | |
CO2 Incubator | Panasonic | KM-CC17RU2 | |
Copper Grids | Electron Microscopy Sciences | G100-Cu | Ultra-thin sections put on the grids; 100 lines/inch square mesh |
Critical Point Dryer | Leica | EM CPD300 | For drying biological samples for SEM applications in absolute acetone |
DAB/AEC chromogen solution mixture  | Sigma Aldrich | AEC101 | Store at +4 °C |
Diamond knife | Diatome | Ultra 45°, 40-US | Use for ultra-thin sections for TEM |
Dimethyl sulfoxide for molecular biology | Biofroxx | 67-68-5 | |
Disposable Plastic Pasteur Pippettes | Nest | ||
DMEM - Dulbecco's Modified Eagle Medium | Gibco | 41966-029 | Store at +4 °C |
Eosin Y Solution Alcoholic | Bright Slide | 2.BS01-105-1000 | |
Epon resin | Sigma | 45359-1EA-F | Epoxy Embedding Medium kit, Store at +4 °C |
Fetal Bovine Serum with Additive Fortifier | Pan Biotech | P30-3304 | Store at +4 °C |
Freezing Solution (FS) | Cellorama | CellO-F | Store at +4 °C |
Glass knife maker | Leica | EM KMR3 | For make glass knives in 8 mm thickness |
Glass knife strips (Size 8 mm x 25.4 mm x 400 mm) | Leica | 7890-08 | Use for ultra- or semi-thin sections for TEM |
Glutaraldehyde Aqueous Solution, EM grade, 25% | Electron Microscopy Sciences | 16210 | Dilute in dH2O to make 2.5% solution and store at +4 °C |
Glycerol solution | Sigma Aldrich | 56-81-5 | Store at -20 C, Dilution :1:100 |
Goat anti-chicken IgY (H+L) Secondary Antibody,Alexa, 647 | Invitrogen | A32933 | Store at RT |
Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 488 | Invitrogen | 35502 | Store at +4 °C, Dilution :1:50 |
Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 550 | Invitrogen | 84540 | Store at +4 °C, Dilution :1:50 |
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 488 | Invitrogen | 35552 | Store at +4 °C, Dilution :1:50 |
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 550 | Invitrogen | 84541 | Store at +4 °C |
Hematoxylin Harris | Bright Slide | 2.BS01-104-1000 | |
HepG2 cells | ATCC | HB-8065 | Store in nitrogen tank |
Human/Rat OV-6 Antibody Monoclonal Mouse IgG1 Clone # OV-6 | R&D Systems | MAB2020 | Store at -20 °C |
Hydrogel | Corning | 354248 | Matrigel, Basement Membrane Matrix High Concentration (HC), LDEV-free, 10 mL, Store at -20 °C |
Hydrogel | Corning | 354234 | Matrigel, Basement Membrane Matrix, LDEV-free, 10 mL, Store at -20 °C |
Hydrogel | ThermoFischer Scientific | A1413201 | Geltrex, LDEV-Free Reduced Growth Factor Basement Membrane Matrix |
Hydrogel | Biotechne, R&D Systems | BME001-01 | Cultrex Ultramatrix RGF BME, Store at -20 °C |
Karnovsky's fixative | %2 PFA, %2.5 Glutaraldehyde in 0.15 M Cacodylate Buffer, 2 mM CaCl2; prepare fresh; use for TEM & SEM samples | ||
L-Aspartic acid | Sigma | 11189-100G | Store at RT |
Lead aspartate solution | Dissolve 40 mg aspartic acid in 10 mL ddH2O and add 66 mg lead nitrate. Solution stabilize at 60 °C and adjust pH to 5; prepare fresh | ||
Lead nitrate | Electron Microscopy Sciences | 17900 | Store at RT |
Leica Confocal Microscope | Leica | DMi8 | |
LSM 700 Laser Scanning Confocal Microscope | Zeiss | ||
Microplate reader | Biotek Synergy | ||
Multipurpose Device (MD) | Cellorama | CellO-M | |
Nuclear-DNA stain | Invitrogen | H3569 | Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg/mL Solution in Water, Store at +4 °C |
Nuclear-DNA stain | ThermoFischer Scientific | 62248 | DAPI solution, Store at +4 °C |
Osmium Tetroxide (OsO4) ,4% | Electron Microscopy Sciences | 19190 | Dilute in dH2O to make 2% solution; store at +4 °C and in airtight container; protect light |
Ov6 antibody | R&D systems | MAB2020 | Store at +4 °C |
Paraformaldehyde (PFA) solution, 4% | Sigma | 1.04005.1000 | Dissolve 4% PFA in dH2O and boil, cool and aliquot; store at -20 °C |
Paraformaldehyde solution 4% in PBS, 1 L | Santa Cruz Biotechnology | sc-281692 | Store at +4 °C |
Phosphate Buffered Saline (PBS), tablets | MP Biomedicals, LLC | 2810305 | |
Post-fixative solution | %2 OsO4, %2.5 Potassium Ferrocyanide in dH2O; prepare fresh | ||
Potassium Ferrocyanide aqueous solution, 5%Â | Electron Microscopy Sciences | 26603-01 | Store at RT |
Primovert - Inverted Bright Field Microscope - ZEISS | Zeiss | Item no.: 491206-0001-000 | |
Round bottom microcentrifuge tubes, 2 mL | Nest | 620611 | |
Scanning Electron Microscopy with STEM attachment | Zeiss | GeminiSEM 500 | We use Inlens Secondary Electron (SE) detector at 2-3 kV for scanning electron micrographs and aSTEM detector at 30 kV for transmission electron micrographs. |
SensiTek HRP Anti-Polyvalent Lab Pack | ScyTek Laboratories | SHP125 | Store at +4 °C |
Sodium Cacodylate Buffer, 0.4 M, pH 7.2 | Electron Microscopy Sciences | 11655 | Dilute in dH2O to make 0.2 M and store at +4 °C |
Sodium/Potassium ATPase alpha 1 antibody [M7-PB-E9] | GeneTex | GTX22871 | Store at -20 °C |
Solution for Immunofluorescence Labeling (S-IF) | Cellorama | CellO-IF | Store at +4 °C |
Solution for Immunohistochemistry (S-IHC) | Cellorama | CellO-P | Store at +4 °C |
Specimen trimming device | Leica | EM TRIM2 | For prepare epon sample block to ultramicrotome |
Sputter coater | Leica | EM ACE200 | Coat the SEM samples with 6 nm gold/palladium for 90 s |
Thiocarbohydrazide (TCH) | Sigma | 223220-5G | Dilute in dH2O to make 0.5% solution and filter with 0.22 µm membrane filter; store at RT; prepare fresh |
Trypan Blue Solution, 0.4% | Gibco | 15250061 | |
Ultra gel super glue | Pattex | PSG2C | For glue polymerized epon block with sample to holder epon block |
Ultramicrotome | Leica | EM UC7 | For prepare high-quality ultra- or semi-thin sections for transmission electron microscopy (TEM) |
Universal Pipette Tips, 10 µL | Nest | 171215-1101 | |
Universal Pipette Tips, 1000 µL | Isolab | L-002 | |
Universal Pipette Tips, 200 µL | Nest | 110919HA01 | |
Uranyl Acetate | Electron Microscopy Sciences | 22400 | Dilute in dH2O to make 2% solution and filter with 0.22 µm membrane filter; keep tightly closed container store at RT |
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