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

Tendon Araştırma ve Mühendisliği için Üç Adımlı Organoid Model

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.

Yazar Spotlight: 3D Organoid Modellerle Tendon Doku Mühendisliğini İlerletmek

İnsan Dermal Fibroblastları Kullanılarak Kendi Kendine Monte Edilmiş Hücre Tabakası Kültürünün ve 3D Tendon/Ligament Benzeri Organoidin Manuel Olarak Üretilmesinin Gösterilmesi

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

Tendon Araştırma ve Mühendisliği için Yetişkin Normal İnsan Dermal Fibroblastlarının 2D Genişlemesi, 2D Stimülasyonu ve 3D Organoid Olgunlaşması

Başlamak için, santrifüj normal insan dermal fibroblastlarını tripsinizlendirir. Süpernatanı santrifüjlenmiş tüpten çıkarın. Pelete önceden ısıtılmış takviye edilmiş DMEM düşük glikoz ortamı ekleyin ve yeniden süspanse etmek için iyice karıştırın.Normal insan dermal fibroblastlarını 10 santimetrelik yapışık bir hücre kültürü kabında plakalayın. Çanağı çapraz bir şekilde hafifçe sallayın. 14 günlük 2D stimülasyondan sonra, hücreler birleştiğinde, ortamı aspire edin ve oluşan hücre tabakasını tabaktan nazikçe ve hızlı bir şekilde ayırmak için bir hücre kazıyıcı kullanın.Eşzamanlı olarak, hücre tabakasını 3B çubuk benzeri bir organoid haline getirin. Normal insan dermal fibroblast organoidlerini 10 santimetre genişliğinde yapışmayan bir Petri kabına aktarın. Uzamayı bir cetvelle ölçün.Daha sonra organoidin bir tarafını bir cımbızla tutun ve organoidi eksenel olarak yaklaşık% 10 oranında uzayana kadar hafifçe gerin. Bir cımbız kullanarak, sabitlemek için organoidin her iki kenarından metal pimleri manuel olarak plastik tabağa bastırın. Son olarak, 3D olgunlaşma için, inkübasyondan önce 10 mililitre önceden ısıtılmış takviye edilmiş DMEM yüksek glikoz ortamı ekleyin. 3D olgunlaşma aşamasının sıfırıncı ve 14. günlerinde 3D organoidlerin brüt morfolojisi parıldayan, beyaz bir görünüm gösterdi.Zamanla, organoidler büzüldü ve daha yoğun göründü. Organoidlerin morfolojisi H E boyaması kullanılarak değerlendirildi. İlk gün, organoidler, esas olarak düşük miktarda matris ile çevrili hücrelerden oluşan bağlantısız katmanlar gösterdi.Katmanlar içindeki hücreler, yuvarlak bir şekle ve değişen boyutlara sahip çekirdekler sergiledi. 14. günde, eozin sinyalinde gözle görülür bir artış gözlendi, bu da matris birikimini ve kaynaşmış katmanları gösterdi. Ayrıca, organoidler hizalanmış hücreler içeren alanların varlığını gösterdi.Çekirdeklerin çoğunluğu benzer boyutlara sahipti ve bazen uzamıştı.

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Research

JoVE Journal

Bioengineering

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

237 Görüntüleme.  University of Würzburg. Başlamak için, santrifüj normal insan dermal fibroblastlarını tripsinizlendirir. Süpernatanı santrifüjlenmiş tüpten çıkarın. Pelete önceden ısıtılmış takviye edilmiş DMEM düşük glikoz ortamı ekleyin ve yeniden süspanse etmek için iyice karıştırın.Normal insan dermal fibroblastlarını 10 santimetrelik yapışık bir hücre kültürü kabında plakalayın. Çanağı çapraz bir şekilde hafifçe sallayın. 14 günlük 2D stimülasyondan sonra, hücreler bir...

Video: Tendon Araştırma ve Mühendisliği için Yetişkin Normal İnsan Dermal Fibroblastlarının 2D Genişlemesi, 2D Stimülasyonu ve 3D Organoid Olgunlaşması

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 arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

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