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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Culturing organoids and spheroids

  1. Place the hydrogel on ice overnight (in a refrigerator or a cold room) to thaw.
  2. Place the commercially available multipurpose device (MD; see Table of Materials) in the incubator 1 day before the experiment (37 °C, 5% CO2) to warm.
  3. Place sterile wide-ended pipette tips in the refrigerator at 4 °C.
    NOTE: Steps 1.1-1.3 are to be performed on Day 0, and steps 1.4-1.11 are to be performed on Day .......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Absolute Ethanol (EtOH)Merck8187602500Dilute in dH2O to make 30%, 50%, 70%, 80%, 90% and 96% solution and store at RT
AcetoneMerck8222512500Store at RT
Alexa fluor wheat germ agglutinin and Hoechst  in Hank's balanced salt solution (HBSS)  InvitrogenI34406Image-IT LIVE Plasma Membrane and Nuclear Labeling Kit, Store at -20 °C
Alpha-1-Fetoprotein (AFP) Concentrated and Prediluted Polyclonal AntibodyBiocare MedicalCP 028 AStore at +4 °C
Anti-albumin antibodyAbcamEPR20195Store at +4 °C, Dilution: 1:50
Anti-beta galactosidase antibody, Chicken polyclonalAbcam134435Store at +4 °C, Dilution: 1:25
Anti-cytokeratin 5Abcam53121Store at +4 °C, Dilution: 1:100
Arginase-1 Concentrated and Prediluted Rabbit Monoclonal AntibodyBiocare MedicalACI 3058 A, BStore at +4 °C, Dilution: 1:50
Calcium chloride (CaCl2)SigmaC1016-500GDissolve in Karnovsky's fixative to make 2 mM CaCl2; store at RT
Cell Counting Kit 8 (WST-8 / CCK8)Abcamab228554
Centrifuge tubes, 15 mL Nest601051
Centrifuge tubes, 50 mL Nest602052
Class II Microbiological Safety Cabinet Bio II Advance PlusTelstarEN12469
CO2 IncubatorPanasonicKM-CC17RU2
Copper GridsElectron Microscopy SciencesG100-CuUltra-thin sections put on the grids; 100 lines/inch square mesh
Critical Point DryerLeicaEM CPD300For drying biological samples for SEM applications in absolute acetone
DAB/AEC chromogen solution mixture  Sigma AldrichAEC101Store at +4 °C
Diamond knifeDiatomeUltra 45°, 40-USUse for ultra-thin sections for TEM
Dimethyl sulfoxide for molecular biologyBiofroxx67-68-5
Disposable Plastic Pasteur PippettesNest
DMEM - Dulbecco's Modified Eagle MediumGibco41966-029Store at +4 °C
Eosin Y Solution AlcoholicBright Slide2.BS01-105-1000
Epon resin Sigma45359-1EA-FEpoxy Embedding Medium kit, Store at +4 °C
Fetal Bovine Serum with Additive FortifierPan BiotechP30-3304Store at +4 °C
Freezing Solution (FS)CelloramaCellO-FStore at +4 °C
Glass knife makerLeicaEM KMR3For make glass knives in 8 mm thickness
Glass knife strips (Size 8 mm x 25.4 mm x 400 mm)Leica7890-08Use for ultra- or semi-thin sections for TEM
Glutaraldehyde Aqueous Solution, EM grade, 25% Electron Microscopy Sciences16210Dilute in dH2O to make 2.5% solution and store at +4 °C
Glycerol solutionSigma Aldrich56-81-5Store at -20 C, Dilution :1:100
Goat anti-chicken IgY (H+L) Secondary Antibody,Alexa, 647InvitrogenA32933Store at RT
Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 488Invitrogen35502Store at +4 °C,  Dilution :1:50
Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight, 550Invitrogen84540Store at +4 °C,  Dilution :1:50
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 488Invitrogen35552Store at +4 °C,  Dilution :1:50
Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight, 550Invitrogen84541Store at +4 °C
Hematoxylin Harris Bright Slide2.BS01-104-1000
HepG2 cellsATCCHB-8065Store in nitrogen tank
Human/Rat OV-6 Antibody Monoclonal Mouse IgG1 Clone # OV-6R&D SystemsMAB2020Store at -20 °C
HydrogelCorning354248Matrigel, Basement Membrane Matrix High Concentration (HC), LDEV-free, 10 mL, Store at -20 °C
HydrogelCorning354234Matrigel, Basement Membrane Matrix, LDEV-free, 10 mL, Store at -20 °C
HydrogelThermoFischer ScientificA1413201Geltrex, LDEV-Free Reduced Growth Factor Basement Membrane Matrix
HydrogelBiotechne, R&D SystemsBME001-01Cultrex 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 acidSigma11189-100GStore at RT
Lead aspartate solutionDissolve 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 nitrateElectron Microscopy Sciences17900Store at RT
Leica Confocal MicroscopeLeicaDMi8
LSM 700 Laser Scanning Confocal MicroscopeZeiss
Microplate readerBiotek Synergy
Multipurpose Device (MD)CelloramaCellO-M
Nuclear-DNA stainInvitrogenH3569Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg/mL Solution in Water, Store at +4 °C
Nuclear-DNA stainThermoFischer Scientific62248DAPI solution, Store at +4 °C
Osmium Tetroxide (OsO4) ,4%Electron Microscopy Sciences19190Dilute in dH2O to make 2% solution; store at +4 °C and in airtight container; protect light
Ov6 antibodyR&D systemsMAB2020Store at +4 °C
Paraformaldehyde (PFA) solution, 4% Sigma1.04005.1000Dissolve 4% PFA in dH2O and boil, cool and aliquot; store at -20 °C
Paraformaldehyde solution 4% in PBS, 1 LSanta Cruz Biotechnologysc-281692Store at +4 °C
Phosphate Buffered Saline (PBS), tabletsMP Biomedicals, LLC2810305
Post-fixative solution%2 OsO4, %2.5 Potassium Ferrocyanide in dH2O; prepare fresh
Potassium Ferrocyanide aqueous solution, 5% Electron Microscopy Sciences26603-01Store at RT
Primovert - Inverted Bright Field Microscope - ZEISSZeissItem no.: 491206-0001-000
Round bottom microcentrifuge tubes, 2 mLNest620611
Scanning Electron Microscopy with STEM attachmentZeissGeminiSEM 500We 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 PackScyTek LaboratoriesSHP125Store at +4 °C
Sodium Cacodylate Buffer, 0.4 M, pH 7.2Electron Microscopy Sciences11655Dilute in dH2O to make 0.2 M and store at +4 °C
Sodium/Potassium ATPase alpha 1 antibody [M7-PB-E9]GeneTexGTX22871Store at -20 °C
Solution for Immunofluorescence Labeling (S-IF)CelloramaCellO-IFStore at +4 °C
Solution for Immunohistochemistry (S-IHC)CelloramaCellO-PStore at +4 °C
Specimen trimming deviceLeicaEM TRIM2For prepare epon sample block to ultramicrotome
Sputter coaterLeicaEM ACE200Coat the SEM samples with 6 nm gold/palladium for 90 s
Thiocarbohydrazide (TCH)Sigma223220-5GDilute 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%Gibco15250061
Ultra gel super gluePattexPSG2CFor glue polymerized epon block with sample to holder epon block
UltramicrotomeLeicaEM UC7For prepare high-quality ultra- or semi-thin sections for transmission electron microscopy (TEM)
Universal Pipette Tips, 10 µLNest171215-1101
Universal Pipette Tips, 1000 µLIsolabL-002
Universal Pipette Tips, 200 µL Nest110919HA01
Uranyl AcetateElectron Microscopy Sciences22400Dilute in dH2O to make 2% solution and filter with 0.22 µm membrane filter; keep tightly closed container store at RT

References

  1. Clevers, H. Modeling development and disease with organoids. Cell. 165 (7), 1586-1597 (2016).
  2. Drost, J., Clevers, H. Organoids in cancer research. Nature Reviews Cancer. 18 (7), 407-418 (2018).
  3. Nath, S., Devi, G. R.

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OrganoidsSpheroidsHydrogel3D CultureFreezingThawingProcessingImagingMicroscopyDisease ModelingBiomarkersHEPG2 CellsImmunofluorescence LabelingFixationWashing

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