Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

Summary

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Abstract

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (> 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Introduction

Although 2D cell culture is widely used in cancer research, limitations exist as the cells are grown in a monolayer format with a uniform concentration of nutrients and oxygen. These cultures lack important cell-cell and cell-matrix interactions present in the native tumor microenvironment (TME). Consequently, these models poorly recapitulate physiological conditions, resulting in aberrant cell behaviors, including unnatural morphologies, irregular receptor organization, membrane polarization, and abnormal gene expression, among other conditions^1,2,3,

Protocol

1. Preparation of the Materials, Hydrogel, and Cell Culture Materials

1. Material and solution preparation
   1. Wash and dry 250 mL and 100 mL glass beakers, magnetic stirrers, spatulas, 10 mL cartridges, 25 G cylindrical nozzles (with a length of 0.5 in and an inner diameter of 250 µm). Sterilize the materials by autoclaving them at 121 °C/15 min/1 atm. Keep the materials under sterile conditions until use.
      Note: Refer to the Table of Materials for vendor information.
   2. Weigh 3 g of alginate (3% w/v) and 7 g of gelatin (7% w/v) (denoted as A3G7 hereafter).
   3. Sterilize the following ....

Results

The temperature sweep shows a distinct difference of the A3G7 precursor at 25 °C and 37 °C. The precursor is liquid at 37 °C and has a complex viscosity of 1938.1 ± 84.0 mPa x s, which is validated by a greater G" over G'. As the temperature decreases, the precursor undergoes physical gelation due to the spontaneous physical entanglement of the gelatin molecules into a tri-helix formation^29,30. Both the G' and the G" increase and .......

Discussion

Cell-laden structures can be compromised if contamination (biological or chemical) occurs at any point in the process. Usually, biological contamination is seen after two or three days of culture as a color change in the culture media or the bioprinted structure. Therefore, the sterilization (physical and chemical disinfection) is a key step for all the cell-related processes. Noteworthy, autoclaving gelatin changes its gelling properties, which made it gel slower in the trials we conducted. Therefore, we sterilized the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium alginate	FMC BioPolymer	CAS-No: 9005-38-3	Protanal LF 10/60 FT
Gelatin	Sigma-Aldrich	G9391	Type B gelatin from bovine skin
Dubelcco's phosphate buffered saline (DPBS 1X)	Gibco	LS14190136	1×, w/o calcium, w/o magnesium
Magnetic hotplate	Corning	N/A	Stirrer/hot plate model PC-420
50 mL centrifuge tubes	Corning	352098	Falcon® 50mL High Clarity PP Centrifuge Tube, Conical Bottom, Sterile
Centrifuge	GMI	N/A	Sorvall RT6000D, GMI, USA
Calcium chloride anhydrous	Sigma-Aldrich	C1016
MilliQ water	Millipore	N/A
Millipore 0.22 µm filters	Millipore	SLGS033SB	Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized
Oscillation rheometer MCR 302	Anton Paar	N/A
Rheometer measuring tool CP25	Anton Paar	79038	Conical plate geometry for rheometer
RheoCompass	Anton Paar	N/A	Software controlling rheometer MCR 302
Scanning electron microscope	Hitachi	N/A	SEM, Hitachi SU-3500 Variable Pressure
Paraformaldehyde, 96%, extra pure	Acros Organics	416785000
Dulbecco modified eagle medium (DMEM)	Gibco	11965092
Antibiotic/Antimycotic solution (100X) stabilized	Sigma	A5955
Fetal bovine serum	Wisent Bioproducts	080-150
Cell culture T-75 flasks	Sigma-Aldrich	CLS430641	75 cm2 TC-Treated surface treatment
3D bioprinter BioScaffolder 3.1	GeSiM	N/A
GeSim software	GeSiM	N/A	Software controlling BioScaffolder 3.1
10cc cartridge UV resist	EFD Nordson	7012126
End cap	EFD Nordson	7014472
Tip cap	EFD Nordson	7014469
Piston	EFD Nordson	7012182
Stainless nozzle G25	EFD Nordson	7018345
Water bath	VWR	N/A
Agarose	Sigma-Aldrich	A9539	Bioreagent, for molecular biology
Costar 6-well plates	Corning	3516	TC-Treated Multiple Well Plates, Individually Wrapped, Sterile
Confocal spinning disk inverted microscope	Olympus Life Science	N/A	Olympus IX83
MTS assay kit	Promega	G3582	CellTiter 96® AQueous One Solution Cell Proliferation Assay
Live/Dead viability cytotoxicity kit	Molecular Probes,ThermoFisher Scientific	L3224
Trypsin 0.25/EDTA 1X	Gibco	25200-072
Corning 96-well plate	Corning	3595	Clear Flat Bottom Polystyrene TC-Treated Microplate, Individually Wrapped, with Low Evaporation Lid, Sterile
Autoclave Tuttnauer	Heidolph Brinkmann	N/A	Heidolph Tuttnauer 2540E Autoclave Sterilizer Electronic Model with 4 Stainless Steel Trays, 23L Capacity
Trypan blue	Invitrogen	T10282	0.4% solution
Ethanol	Commercial Alcohols	P016EA95	Greenfield Speciality Alcohols
CO2 Incubator	Panasonic	N/A	MCO 19AIC-PA
Lyophilizer	SP Scientific	N/A	Virtis Sentry 2.0
SolidWorks	Dassault Systems	N/A	A CAD software used to build demostrative propeller-like model
MATLAB	The MathWorks	N/A	A programming software used to generate G-code for BioScaffolder 3.1