2d expansion, 2d stimulation, and 3d organoid maturation of adult normal human dermal fibroblasts for tendon research and engineering

Three-Step Organoid Model for Tendon Research and Engineering

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

मानव त्वचीय फाइब्रोब्लास्ट का उपयोग करके स्व-इकट्ठे सेल शीट संस्कृति और 3 डी कण्डरा / स्नायुबंधन जैसे अंग के मैनुअल जनरेशन का प्रदर्शन

2 डी विस्तार, 2 डी उत्तेजना, और कण्डरा अनुसंधान और इंजीनियरिंग के लिए वयस्क सामान्य मानव त्वचीय फाइब्रोब्लास्ट की 3 डी ऑर्गेनॉइड परिपक्वता

शुरू करने के लिए, अपकेंद्रित्र ने सामान्य मानव त्वचीय फाइब्रोब्लास्ट को ट्रिप्सिनाइज्ड किया। अपकेंद्रित्र ट्यूब से सतह पर तैरनेवाला निकालें. गोली के लिए prewarmed पूरक DMEM कम ग्लूकोज माध्यम जोड़ें, और अच्छी तरह से मिश्रण यह resuspend है.एक 10 सेंटीमीटर पक्षपाती सेल संस्कृति पकवान में सामान्य मानव त्वचीय fibroblasts प्लेट. धीरे से डिश को क्रॉसवर्ड तरीके से रॉक करें। 2 डी उत्तेजना के 14 दिनों के बाद, जब कोशिकाओं को संगम हो गया है, मध्यम महाप्राण, और धीरे और जल्दी से पकवान से गठित सेल शीट अलग करने के लिए एक सेल खुरचनी का उपयोग करें.इसके साथ ही, सेल शीट को 3 डी रॉड जैसे ऑर्गेनॉइड में रोल करें। सामान्य मानव त्वचीय फाइब्रोब्लास्ट ऑर्गेनोइड को 10 सेंटीमीटर चौड़े गैर-पक्षपाती पेट्री डिश में स्थानांतरित करें। एक शासक के साथ बढ़ाव को मापें।फिर एक चिमटी के साथ ऑर्गेनॉइड के एक तरफ पकड़ो, और धीरे से ऑर्गेनॉइड को तब तक फैलाएं जब तक कि यह अक्षीय रूप से लगभग 10% तक लम्बा न हो जाएएक चिमटी का उपयोग करके, इसे ठीक करने के लिए ऑर्गेनॉइड के दोनों किनारों के माध्यम से धातु के पिन को प्लास्टिक डिश में मैन्युअल रूप से दबाएं। अंत में, 3 डी परिपक्वता के लिए, इनक्यूबेशन से पहले से गर्म पूरक डीएमईएम उच्च ग्लूकोज माध्यम के 10 मिलीलीटर जोड़ें। 3 डी परिपक्वता चरण के शून्य और 14 दिनों में 3 डी ऑर्गेनोइड की सकल आकृति विज्ञान ने एक चमकदार, सफेद उपस्थिति दिखाई।समय के साथ, ऑर्गेनोइड सिकुड़ गए और सघन दिखाई दिए। ऑर्गेनोइड की आकृति विज्ञान का मूल्यांकन एचई धुंधला का उपयोग करके किया गया था। पहले दिन, ऑर्गेनोइड ने डिस्कनेक्ट की गई परतों को दिखाया, मुख्य रूप से कम मात्रा में मैट्रिक्स से घिरे कोशिकाओं से बना था।परतों के भीतर की कोशिकाओं ने एक गोल आकार और अलग-अलग आकार के साथ नाभिक का प्रदर्शन किया। 14 वें दिन, ईोसिन सिग्नल में ध्यान देने योग्य वृद्धि देखी गई, जिसने मैट्रिक्स जमाव और फ्यूज्ड परतों का संकेत दिया। इसके अलावा, ऑर्गेनोइड ने संरेखित कोशिकाओं वाले क्षेत्रों की उपस्थिति दिखाई।अधिकांश नाभिकों के समान आकार थे, और कभी-कभी लम्बी होती थीं।

Research

JoVE Journal

Bioengineering

2D Expansion, 2D Stimulation, and 3D Organoid Maturation of Adult Normal Human Dermal Fibroblasts for Tendon Research and Engineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...