Generating Free-floating Normal Human Epithelial-Fibroblast Spheroid Co-Cultures

Summary

Here, we present a simple and very practical but reliable protocol for direct spheroidal co-cultivation of human lung epithelial cells and fibroblasts without relying on a matrix.

Abstract

Three-dimensional (3D) culture systems have become increasingly popular due to their ability to reproduce natural cell properties and architectures, thus mimicking tissue-like structures in vitro. Among these models, the culture of spherical cell aggregates (so-called spheroids) embedded in a semi-solid extracellular matrix (ECM) represents an advanced near-in vivo cell culture model, as it allows for different functional cell states as a result of cell-cell and cell-ECM interactions, as well as oxygen and nutrient gradients. Because spheroids are technically less demanding and relatively inexpensive to obtain, they are frequently used in drug screening and toxicity testing, enabling high-content screening for testing new drugs in the preclinical phase. For this purpose, 3D structures of various tumors are predominantly recreated. At the same time, Matrigel is currently one of the most widely used ECMs and is considered the gold standard in spheroid and organoid cultivation, although this is a 3D matrix based on mouse experiments, the extraction of which- like animal testing in general-is associated with major ethical concerns. Here, we present a matrix-free protocol for direct spheroidal co-cultivation of human bronchial epithelial cells and fibroblasts, which can be considered as an optimized co-cultivation method, especially concerning epithelial-fibroblast communication and interactions within the respective spheroids, without relying on an (animal-based) carrier matrix. By establishing these free-floating epithelial-fibroblastoid spheroid co-cultures as a patient-oriented in vitro platform to model normal (lung) tissue toxicities of cancer therapeutic approaches, not only a reduction in the number of experimental animals but also an adequate and meaningful replacement of corresponding animal experiments can be achieved.

Introduction

Two-dimensional (2D) cell cultures, in which flat monolayer cells are cultured, expanded, and then plated for cell-based research assays, remain an easy, convenient, cost-effective, and widely used method for standard preclinical screening procedures^1,2. To study the interactive (paracrine) crosstalk between various types of cells in vitro, a conditioned medium from one cell type for stimulation of another cell type can be used. Additionally, various co-culture methods emerged, e.g., indirect methods, when co-cultured cells are physically separated using a microporous membrane. These 'Transwell sy....

Protocol

All steps are performed under sterile conditions (Biosafety cabinet). For the media compositions used in this study, refer to Table 1.

1. Preparation of experimental material

1. Culture and expand human normal lung epithelial cells (HBEC3-KT) on standard plastic cell culture dishes (T75 flasks) in normal epithelial growth medium using a standard incubator¹³ (see Table 1).
   NOTE: For improved growth, the medium can be additionally supplemented with 10% fetal calf serum (FCS).
2. Culture human fibroblasts (HS-5) in RPMI Medium supplemented with 10....

Results

This simple protocol enables the matrix-free spheroidal co-cultivation of human bronchial epithelial cells and fibroblasts in combination with ultra-low attachment plates (Figure 1A). During the cultivation process, more complex lung spheroids gradually form from the initially generated spheroids, with the efficient generation of different epithelial and fibroblastic structures representing direct cellular interactions as revealed by morphological analyses using live cell imaging (

Discussion

3D cell culture models have been proposed to bridge the gap between 2D cultures and in vivo studies, as this type of (co-) culture better mimics the physiological, morphological, and pathological properties present in vivo because more in vivo-like cell-cell interactions, cell-ECM interactions, responses to stimuli, gene expression, protein expression, and differentiation are present^17,18. In addition to the numerous and sometimes very .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL sterile serological pipettes	ThermoFischer	170356N	Nunc sterile serological pipettes, or equivalent
15 mL conical tube	Corning	430052	or equivalent
37 °C/ 5% CO2 humidified  incubator
5 mL sterile serological pipettes	ThermoFischer	170355N	Nunc sterile serological pipettes, or equivalent
50 mL conical tube	Corning	CLS430829	or equivalent
ATRA (All-Trans Retinoic Acid)	StemCell Technologies	Cat#72262	50 nM
B-27 Supplement (50x),	ThermoFischer/Gibco	Cat#17504044	1x
BD Rhapsody WTA Amplification Kit	BD Bioscience	Cat#633801	use according to the manufacturer's instructions
Biosafety cabinet
Bovine Serum Albumin	Roche	Cat#BSAVHS-RO	0.4% (v/v)
Cell Strainer	Corning	352350	Falcon 70 µm Cell Strainer, White, Sterile, Individually Packaged
Centrifuge	Eppendorf	https://www.fishersci.com/shop/products/eppendorf-5804-series-centrifuge-rotor-packages-9/p-4119001	e.g., Eppendorf Centrifuge 5804 - Benchtop Centrifuge
DMEM/F12	Invitrogen	Cat#11330-032	500 mL
DNase I, RNase-free (1 U/μL)	ThermoFischer	Cat#EN0521	100 U/mL
EDTA (0,5 M), pH 8,0, RNase-free	ThermoFischer	AM9260G	2 mM
Eppendorf Research plus 12 channel multichannel pipette	Eppendorf		e.g., #3125000044 (10–100 µL) or #3125000060 (30–300 µL)
Fetal calf serum	ThermoFischer/Gibco	Cat#A5256701	10% (v/v)
HBEC3-KT	ATCC	CRL-4051	human normal lung epithelial cell line
Heparin Solution (0.2%)	StemCell Technologies	Cat#7980	0.8 mL
Hoechst 33342	ThermoFischer	Cat#H1399	1 µg/mL
HS-5	ATCC	CRL-3611	human stromal fibroblasts
Hu Recom FGF-10 (KGF-2) ACF	StemCell Technologies	Cat#78173.1	10 ng/mL
Hu Recom FGF-7 (KGF) ACF	StemCell Technologies	Cat#78186.1	10 ng/mL
Human Single-Cell Multiplexing Kit	BD Bioscience	Cat#633781	use according to the manufacturer's instructions
Hydrocortisin Stock Solution	StemCell Technologies	Cat#7926	0.5 mL per 500 mL
Laduviglusib (CHIR-99021)	Biozol/Selleckchem	Cat#S1263	3 µM
L-Ascorbic acid	Sigma-Aldrich	Cat#A92902	50 µg/mL
Microscope	ZEISS
Monothioglycerol	Sigma-Aldrich	Cat#M6145	0.4 µM
N-2 Supplement (100x)	ThermoFischer/Gibco	Cat#17502048	1x
Neubauer counting chamber	Merck	BR718605	e.g., BRAND counting chamber BLAUBRAND Neubauer pattern
Normal goat serum	ThermoFischer	Cat#31873	2 % (v/v) in PBS
NP-40	Merck/Millipore	Cat#492016	NP-40-Alternative, 1 %
PAS Staining Kit	Sigma Aldrich	Cat#1016460001	use according to the manufacturer's instructions
PBS	ThermoFischer	Cat#10010023	1x
Penicillin-Streptomycin (10.000 U/mL), 100x	ThermoFischer/Gibco	Cat#15140122	1x
PFA	ThermoFischer	Cat#J61899.AK	Paraformaldehyd, 4 % in PBS
PneumaCult Airway Organoid Differentiation Medium (ODM)	StemCell Technologies	Cat#05060	PneumaCult Airway Organoid Basal Medium (#05061, 360 mL) and PneumaCult Airway Organoid Differentiation Supplement* (#05063, 40 mL)
PneumaCult Ex Plus Medium	StemCell Technologies	Cat#5040	Kit consisting of PneumaCult-Ex Plus Basal Medium (#05041, 490 mL ) and PneumaCult-Ex Plus 50X Supplement*(#05042, 10 mL)
Protease inhibitor cocktail	Merck/Roche	Cat#4693132001	1 tab/10 mL
Rhapsody cDNA Kit	BD Bioscience	Cat#633773	use according to the manufacturer's instructions
RPMI 1640 Medium	ThermoFischer/Gibco	Cat#11875093	500 mL
Sodium Chloride	Merck/Sigma-Aldrich	S9625	150 mmol/L
Sodium Citrate	Merck/Sigma-Aldrich	Cat#1613859-1G	0.10%
Sodium Deoxycholate	Merck/Sigma-Aldrich	Cat#30970	0.50%
Sodium Dodecyl Sulfate	Merck/Sigma-Aldrich	Cat#11667289001	0.10%
Sterile reagent reservoir for multichannel micropipette (e.g., 60 ml)	ThermoFischer	9510037	or equivalent
TC-Platte 96well BIOFLOAT a 4 Stück	Sarstedt (or facellitate)	83.3925.400 (or F202003)	ultra-low attachment plate
Tris/HCl	ThermoFischer	Cat#AM9855G	50 mmol/L , pH 8
Triton X-100	Sigma-Aldrich	Cat#X100-5ML	0.05 %
Trypan Blue Stain (0.4%)	ThermoFischer	Cat#15250061	1/5 (v/v) with NGM
TrypLE	ThermoFischer	Cat#12604013	1x
Trypsin-EDTA (0.5 %)	ThermoFischer	Cat#15400054	0.05 % Trypsin-EDTA (TE) solution aseptically diluted to 1X using phosphate buffered saline (without calcium and magnesium)
WST-1 reagent	CELLPRO-RO, Roche	Cat#5015944001	cell proliferation reagent