3D Cell Culture-Based Hybrid Bioanalytical Platform for Optical Imaging Utilizing Silk Fibroin Sponges

Summary

The article describes a simple, accessible, and reproducible methodology for silk scaffold production and culture of 4T1-iRFP720 breast cancer cells under flow-controlled conditions to support dynamic culture. Cellular growth and migration were evaluated as proof of concept using an in vivo optical imaging instrument as the main non-invasive readout.

Abstract

We present an innovative bioanalytical hybrid platform designed for the preclinical evaluation of cellular characteristics. The system combines a three-dimensional (3D) cell culture grown on an artificial extracellular matrix with a chromatography-inspired array configuration. Sponges, made from the structural protein silk fibroin, serve both as a biomimetic extracellular matrix and as a stationary phase. Silk fibroin sponges were produced in-house using a multistep process involving removal of inherent sericin proteins from raw silk fibers, followed by dissolution and dialysis to purify the fibroin solution, dissolution in organic solvent, and subsequent salt-bed casting to generate silk-based sponges with controlled porosity/pore sizes of 500–800 µm. Genetically modified breast cancer cell lines 4T1-iRFP720 and 4T1-wt (non-fluorescent control) were cultured within silk scaffolds using a continuous media flow via a pump, and their cellular growth and characteristics were analyzed non-invasively using optical imaging techniques (in vivo optical imaging instrument). By merging key advantages of chromatographic systems (automatization, reproducibility) with the biological relevance of advanced 3D cell cultures, the platform enables in vitro modeling of tissue-like architecture and morphology while facilitating the monitoring of dynamic cellular behavior. In parallel, the application of medical imaging technology enables real-time and prolonged monitoring of cellular migration and growth, among other factors. This approach offers substantial potential for investigating cellular behaviors at a macroscopic scale in a laminar-like flow system. By improving the physiological relevance of in vitro models, this method may help bridge the translational gap to in vivo studies and is consistent with the reduce, replace, refine (3R) framework for animal experimentation.

Introduction

In living tissues, cell migration and growth occur within a three-dimensional, heterogeneous extracellular matrix (ECM), guided by biophysical and biochemical cues, such as interstitial fluid flow and cell-cell interactions. These factors regulate cell polarity, mechanotransduction, and coordinated movement, underpinning key biological processes including tissue repair and cancer invasion^1,2,3,4.

Despite their importance, most in vitro migration assays, such as scratch and Transwell assays, evaluate singl....

Protocol

The first section outlines the preparation of silk fibroin scaffolds based on prior published work¹⁶. The subsequent sections describe optimization of cell seeding density, integration of flow and optical imaging for monitoring cell movement and growth. This study exclusively utilized established murine breast cancer cell lines (4T1-wt and 4T1-iRFP720) and did not involve human participants, patient samples, primary tissues, or live animals. In accordance with institutional and national guidelines, ethical approval was not required. A schematic overview of the workflow is shown in Figure 1.

Results

This protocol describes a user-friendly, low-cost platform for macroscopic observation of cell growth and movement in a physiologically relevant 3D environment. The method uses silk fibroin sponges as an ECM mimic and incorporates controlled interstitial flow to model dynamic tissue conditions. A schematic overview of step-by-step procedures from silk sponge and scaffold fabrication to cell seeding and experimental platform assembly is depicted in Figure 1.

At the.......

Discussion

The silk sponge production process described in this protocol is standardized, ensuring reproducible scaffold fabrication, as reported in a prior study¹⁶. Production of sponges under fully standardized conditions, such as salt-bed height/amount, silk solution volume, and methanol incubation, is crucial to obtain uniform, comparable sponges. The produced sponges need to follow the quality control protocol criteria published earlier¹⁶. In short terms, the sponge needs to fulf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,1,1,3,3,3-Hexafluoroisopropanol (HFIP)	Sigma-Aldrich	8.04515	Caution: Hazardous. Used for dissolution of freeze-dried silk
24-Well plate	Cellstar, Greiner Bio-One	662102	Storage of cell-seeded scaffolds during incubation
4T1-iRFP720 murine triple-negative breast cancer cells	Macromolecular Cancer Therapeutics Laboratory (MMCT), University of Vienna	N/A	Cell culture experiments; 4T1-iRFP720 was derived in-house from parental 4T1 (ATCC: CRL-2539)
4T1-wt murine triple-negative breast cancer cells	Macromolecular Cancer Therapeutics Laboratory (MMCT), University of Vienna	ATCC: CRL-2539	Cell culture experiments
Bruker Alpha Compact FTIR with Platinum-ATR Sampling Module	Bruker	A220/D-01	FT-IR analysis
Calcium chloride, anhydrous, ≥93% reagent grade	Sigma-Aldrich	902179	For the preparation of dissolution matrix
Cell culture flask, T25	Cellstar, Greiner Bio-One	690175	Consumables; for cell culturing
Cell culture flask, T75	Cellstar, Greiner Bio-One	658175	Consumables; for cell culturing
Centrifuge 5804 R	Eppendorf	5805000010	Used for sample separation by centrifugation
Combi-stopper (female Luer-lock connector)	Braun Melsungen AG	4495101	Used for the platform assembly
Cucurbitacin E	Sigma-Aldrich	SML0577	Cell migration inhibitor
Dermal biopsy puncher, ø 6 mm	Dahlhausen	1190001006	Used to obtain cylindrical sponge scaffolds
Dialysis tubes (3.5 kDa, cellulose)	Spectra/Por by Spectrum Labs	SPEC132724	For dialysis of silk fibroin solution
Dulbecco's Phosphate Buffered Saline	Sigma-Aldrich	D8537	For maintenance of cells
Ethanol, absolute	Merck	1.07017	For the preparation of dissolution matrix
Excel	Microsoft	v2604	Data organization, basic calculations, and plotting
Fetal calf serum (FCS) heat inactivated	Biowest	S181H-500	Supplement for cell culture media
Fiji (former ImageJ)	Open soure (https://imagej.net/software/fiji/)	N/A	Image processing and analysis
GraphPad Prism	GraphPad Software	v8.2.1	Statistical analysis and graph generation
HPLC pump Hitachi L-7120 LaChrom	Merck	K-5244	For dynamic culture conditions in the assembled platform
IVIS 200 Spectrum CT system	PerkinElmer	128201	Optical imaging system with CT capability
IVIS Living Image Software	PerkinElmer	v4.5.2	Software for acquisition, visualization, and quantitative analysis of IVIS imaging data
JEOL JSM-6510 Scanning electron microscope	JEOL GmbH	JSM-6510	SEM images
L-glutamine, 200 mM solution, suitable for cell culture (L-Glu)	Sigma-Aldrich	G7513	Supplement for cell culture media
Lyophilizer Alpha 2-4 LD Plus	Martin Christ	101542	For silk fibroin lyophilization
Male Luer-lock connector	Supelco (Sigma-Aldrich)	57020-U	Adapter for sample reservoirs
Methanol, ≥99.9 %, HPLC Gradient Grade	Carl Roth	7342.1	Caution: Toxic. Induction of beta-sheet formation in silk composites
Optically transparent Solid Phase Extraction (SPE) tubes without frits	Sigma Aldrich	57240-U	Used for the platform assembly
OPUS Spectroscopy software	Bruker	v7.5	FT-IR analysis
Orbital and Linear Shaker MI0103002	Four E'S Scientific	MI0103002	Uniform distribution/mixing
Parafilm PM996	Sigma-Aldrich	P7793	For sealing/wrapping plates and containers for storage (e.g., refrigeration)
Penicillin-Streptomycin, liquid, suitable for cell culture (P/S)	Sigma-Aldrich	P0781	Supplement for cell culture media
Petri dish, 9 cm	VWR	391-0598	For salt-bed sponge casting; for seeding on silk scaffolds
Polyethylene frits for 1 mL SPE tubes	Supelco (Sigma Aldrich)	57244	Used for the platform assembly
RPMI-1640 Medium, suitable for cell culture	Sigma-Aldrich	R0883	Supplemented media is used for silk sponge equilibration, cell culture maintenance and related experiments
Serological pipette, 10 mL	Sarstedt	86.1254.025	Consumables; for maintenance of cells
Serological pipette, 25 mL	Sarstedt	86.1685.020	Consumables; for maintenance of cells
Serological pipette, 5 mL	Sarstedt	86.1253.025	Consumables; for maintenance of cells
Silk cocoons of Bombyx mori L.	Sericulture and Agriculture Experimental Station, Vratsa, Bulgaria	N/A	Raw material for the silk fibroin sponge production
Sodium carbonate, anhydrous, ≥99.5% ACS	VWR	11552.A3	Used for chemical degumming, i.e. removal of sericin
Sodium chloride, ≥98%, technical	VWR	27788.366	For salt-bed sponge casting
SPE Column Push Rod	Carl ROTH	36P9.1	Used for the platform assembly
Stainless steel analytical sieves, 500 µm (ISO 3310-1)	ATECHNIK GmbH	200.050.222-046	For NaCl sieving
Stainless steel analytical sieves, 800 µm (ISO 3310-1)	ATECHNIK GmbH	200.050.222-051	For NaCl sieving
Sterile injectomat line (Tubing), 150 cm, PE	Fresenius Kabi	9004132	Used for dynamic culture conditions
Trypsin - EDTA solution	Sigma-Aldrich	T3924	For maintenance of cells
Ultra-High Performance Centrifuge tubes, 15 mL, sterile	VWR	525-0605	Consumables; for centrifugation, freezing, storage purposes
Ultra-High Performance Centrifuge tubes, 15 mL, unsterile	VWR	525-1083	Consumables; for centrifugation, freezing, storage purposes
Ultra-High Performance Centrifuge tubes, 50 mL, sterile	VWR	525-0609	Consumables; for centrifugation, freezing, storage purposes
Ultra-High Performance Centrifuge tubes, 50 mL, unsterile	VWR	525-1098	Consumables; for centrifugation, freezing, storage purposes
Urine sample cup	Brand	758905	Container for silk solution preparation
Water purification system LaboStar 10 RO DI	Evoqua Water Technologies LLC	W3T324493	Source of ddH2O