A subscription to JoVE is required to view this content. Sign in or start your free trial.
We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.
The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.
Joint diseases like osteoarthritis (OA) are highly prevalent and debilitating and represent a leading cause of disability worldwide1. It is estimated that in the US alone, OA affects 27 million patients and occurs in 12.1% of adults aged 60 and above2. Unfortunately, most drugs currently used to manage joint diseases are palliative, and no effective disease-modifying OA drugs (DMOADs) are available3. This unmet medical need primarily stems from the absence of an effective model for studying the disease mechanisms and developing potential DMOADs. The conventional two-dimensional (2D) cell culture d....
The following protocol follows the ethical guidelines of the University of Pittsburgh and the human research ethics committee of the University of Pittsburgh. Information on the materials used in this study is listed in the Table of Materials.
1. Manufacturing 3D-printed bioreactors
All the tissues of the miniJoint were collected to analyze their phenotypes following 28 days of culture in the miniJoint (Figure 4A). This has been detailed in our previous publication7.
Through the use of RT-qPCR, immunostaining, and histological staining, it was confirmed that the tissue-specific phenotypes were well maintained for the individual microtissues (Figure 4). For example, the osseous component of.......
In this article, we present a protocol for creating a knee joint-on-a-chip system, in which bone, cartilage, adipose tissue, and synovium-like tissues are formed from MSCs and co-cultured within a customized bioreactor. This multi-component, human cell-derived system with plug-and-play features represents a new tool for studying the pathogenesis of joint diseases and developing drugs.
Given that different tissues favor specific culture media, it is critical to provide the respective medium fo.......
This research was primarily supported by funding from the National Institutes of Health (UG3/UH3TR002136, UG3/UH3TR003090). In addition, we thank Dr. Paul Manner (University of Washington) for providing the human tissue samples and Dr. Jian Tan for their help in isolating the MSCs and creating the cell pool.
....Name | Company | Catalog Number | Comments |
3-isobutyl-1-methylxanthine | Sigma -Aldrich | I17018-1G | |
6 well non-tissue culture plate | Corning Falcon® Plates | 351146 | |
24 well non-tissue culture plate | Corning Falcon® Plates | 351147 | |
30 mL syringes | BD Syringe Luer Lock Cascade Health | 302832 | |
Alcian blue stain | EK Industries | 1198 | 1% w/v, pH 1.0 |
Advanced DMEM | Gibco | 12491-015 | |
αMEM | Gibco | 12571-063 | |
Antibiotic-antimycotic | Gibco | 15240-062 | |
Biopsy punch | Integra Miltex | 12-460-407 | |
BODIPY® fluorophore | Molecular Probes | ||
Bone morphogenic protein 7 (BMP7) | Peprotech | ||
Curved forceps | Fisher Brand | 16100110 | |
DMEM | Gibco | 11995-065 | Dulbecco’s Modified Eagle Medium |
Dexmethasome | Sigma -Aldrich | 02-05-2002 | |
E-Shell 450 photopolymer in | EnvisionTec | RES-01-4022 | |
Fetal Bovine Serum | Gemini-Bio Products | 900-208 | |
GlutaMAX | Gibco | 3505-061 | |
gelatin from bovine skin | Hyclone | 1003372809 | |
Hank’s Balanced Salt Solution | Sigma -Aldrich | SH30588.02 | |
indomethacin | Sigma -Aldrich | I7378-56 | |
Insulin-Transferrin-Selenium-Ethanolamine (ITS) | Gibco | 51500-056 | |
interleukin 1β | Peprotech | 200-01B | |
Leur-loc connectors | Cole-Parmer Instruments | 45508-50 | |
L-proline | Sigma -Aldrich | 115388-93-7 | |
β-glycerophosphate | Sigma -Aldrich | 1003129352 | |
Medium bags | KiYATEC | FC045 | |
Methacrylic Anhydride | Sigma -Aldrich | 102378580 | |
Phosphate buffered Saline | Corning | 21-040-CM | |
Pointed forceps | Fisher Brand | 12000122 | |
Silicon mold | McMaster-Carr | RC00114P | |
Silicon o-rings | McMaster-Carr | ZMCCs1X5 | 1mm x 5mm |
SolidWorks | Dassault Systèmes SE, Vélizy-Villacoublay, France | ||
Surgical Blades | Integra Miltex | 4-122 | |
Syringe pump | Lagato210P, KD Scientific | Z569631 | 10 syringe racks |
T-182 tissue culture flasks | Fisher Brand | FB012939 | |
Tissue Culture Dish 150 mm | Fisher Brand | FB012925 | |
Transforming Growth Factor Beta (TGF-β3) | Peprotech | 100-36E | |
Trypsin | Gibco | 25200-056 | |
UV Flashlight | KBS | KB70109 | 395 nm |
Vida Desktop 3D Printer | EnvisionTec | ||
Vitamin D3 | Sigma -Aldrich | 32222-06-3 | 1,25-dihydroxyvitamin D3 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved