Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Summary

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Abstract

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Introduction

A major obstacle in the study of fibrotic diseases is the lack of representative human 3D tissue models that provide insight into the behavior of fibroblasts and their pathological derivatives. To study fibrotic processes, standard 2D culture systems are sub-optimal since isolated fibroblasts transdifferentiate rapidly into α-smooth muscle actin (SMA)-expressing myofibroblasts when cultured on non-compliant 2D substrates^1,2,3. Thus, fibroblasts in the standard 2D culture do not reflect a regular "healthy" tissue phenotype^3,....

Protocol

All steps must be undertaken in a Class II biosafety hoods installed in laboratories under containment level 1. Depending on local regulations and type of manipulations to be performed, such as viral-mediated gene transfer, the containment level must be increased to the biosafety level 2 or 3. All cultures are maintained at 37 °C in a cell culture incubator with a humidified atmosphere of 5 % CO₂ in the air. Note that the volumes (Steps 1 and 2) are provided for a T75 cell culture flask. Adjust the volumes to different culture formats according to the standard cell culture recommendations.

1. Thawing and pre-plating primary cardiac....

Results

ECT reach around 95 % compaction compared to the initial cell-collagen hydrogel volume within the first 24 h. Tissue compaction and contraction under control conditions and in the presence of FCS ensues a few hours after casting and notably increases up to day 5 (Figure 5A). Pole deflection may further increase during the following 15 days (20 days was the longest time tested). The magnitude of pole deflection depends on cell type, cell state, and cell and tissue culture conditions. Typicall.......

Discussion

The presented protocol describes the generation of ECT from primary human CF, which allows studying the mechanical impact of these cells on their extracellular matrix environment and vice-versa.

The fibroblasts need to be expanded to yield sufficient cells for the planned ECT experiments (0.75 x 10⁶ cells/ECT). For the best reproducibility, it is advised to pre-culture frozen or tissue-derived fibroblasts in 2D monolayer culture for a standardized duration up to 80 % confluency with.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture reagents:
Accutase Solution	Merk Millipore	SCR005
Dissociation reagent – TrypLE Express	Gibco	12604013
Dulbecco's Modified Eagle Medium (DMEM) powder, high glucose	Gibco	12100061
Dulbecco’s phosphate buffered saline (DPBS), pH 7.2, -Ca2+, -Mg2+	Gibco	14190144
FGM-2 Fibroblast Growth Medium-2 BulletKit	Lonza	CC-3132
FBM Fibroblast Growth Basal Medium	Lonza	CC-3131
FGM-2 Fibroblast Growth Medium-2 SingleQuots, Supplements and Growth Factors	Lonza	CC-4126
Fibroblast Growth Medium 3 KIT	PromoCell	C-23130
Fibroblast Basal Medium 3	PromoCell	C-23230
Growth Medium 3 SupplementPack	PromoCell	C-39350
Penicillin (10000 U/mL)/ Streptomycin (10000 μL/mL)	Gibco	15140122
Sodium hydroxide solution (NaOH) 1.0 N	Sigma-Aldrich	S2770-100ML
Cell sources:
Normal human cardiac fibroblasts from the ventricle (NHCF-V)	Lonza	CC-2904
Human Cardiac Fibroblasts (HCF-c)	PromoCell	C-12375
Human Cardiac Fibroblasts (HCF-p)	PromoCell	C-12377
Primary human foreskin fibroblasts-1 (HFF-1)	ATCC	SCRC- 1041
Collagen sourses:
Collagen Type I (bovine) in 0.01 M HCl	LLC Collagen Solutions	FS22024	6-7 mg/mL
Collagen Type I (rat tail) in 0.02 M HCl	Corning	354236	~4 mg/mL
Drugs:
Latrunculin-A (Lat-A)	Enzo Life Sciences	BML-T119-0100
Plastic ware:
Cell culture plastic ware	Sarstedt and Starlab
Mesh cell strainer (Nylon, pore size 40 μm)	Falcon	352340
myrPlate-uniform	myriamed GmbH	TM5 med
Serological pipettes wide opening, sterile (10 mL)	Corning	07-200-619
Specific instruments:
Bi-telecentric CORE lens for 1/2″ detectors	OptoEngineering	TCCR12096
Area scan camera Basler ace acA4024	Basler	107404