Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel

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 cultures^1,2,3. 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 diseases^4,5,6,7,8,9. 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 1.
4. Place the hydrogel on ice in a laminar flow hood for 15 min.
   1. Optional: Dilute the hydrogel in cold cell culture medium accord....

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.......

Materials

List of materials used in this article

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 mL	Nest	601051
Centrifuge tubes, 50 mL	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%	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