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

Summary

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.

Abstract

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type 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 TGFß 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.

Introduction

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 disorders^3,4,5. 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 types^{7,8,9,10,11,12,13}. 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 types^14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach^9,16. 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 fibroblasts^7,17,18. 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 (TGFß3), 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 ECM^20,21.

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.

Protocol

NOTE: All the steps must be conducted using aseptic techniques.

1. Culture and pre-expansion of NHDFs

1. Rapidly thaw the cryo-vial containing adult cryopreserved normal human dermal fibroblasts (NHDFs, 1 x 10⁶ cells) at 37 °C until they are almost defrosting.
2. Slowly add 1 mL of prewarmed fibroblast growth medium 2 (ready-to-use kit including basal medium, 2% fetal calf serum (FCS), basic fibroblast growth factor (bFGF) and insulin) supplemented with 1% penicillin/streptomycin (pen/strep) (NHDF medium), prewarmed at 37 °C to the cells.
3. Transfer the cells to a 15 mL centrifuge tube. Centrifuge the cells at 300 x g for 5 min at room temperature (RT).
4. Remove the supernatant. Resuspend the cell pellet in 12 mL of prewarmed NHDF medium.
5. Transfer the cells to a T-75 flask and shake briefly in a cross-wise manner. Place the T-75 flask at 37 °C in a 5% CO₂ humidified incubator.
6. Change the medium every 2 days. Observe the NHDFs under a microscope until they reach 70% - 80% confluency.

2. 2D expansion

1. Take out the T-75 flask of the incubator and remove the NHDF medium. Wash the cells with prewarmed phosphate buffer saline (PBS).
2. Add 3 mL of 0.05% trypsin-EDTA to the cells. Incubate the cells at 37 °C in a 5% CO₂ humidified incubator until the cells detach from the flask (approximately 3 min).
3. Add 6 mL of prewarmed NHDF medium to neutralize the trypsin action. Transfer the cells to a 15 mL centrifuge tube.
4. Centrifuge the cells at 300 x g for 5 min at RT and remove the supernatant.
5. Resuspend the cell pellet in Dulbecco's Modified Eagles Medium (DMEM) low glucose supplemented with 10% fetal bovine serum (FBS), 1x MEM amino acids, 1% pen/strep, prewarmed at 37 °C.
6. Plate the NHDFs in a 10 cm adherent cell culture dish at a density of 8 x 10³ NHDFs/cm² (in total, 4.4 x 10⁵ NHDFs per 10 cm dish). Gently rock in a cross-wise manner.
7. Place the 10 cm cell culture dish at 37 °C in a 5% CO₂ humidified incubator. Change the medium every 2 days
8. Monitor the cells under a microscope until reaching 100% confluency (ca. 5 days).

3. 2D stimulation

1. Take out the 10 cm cell culture dishes containing the NHDFs (from steps 2.6 and 2.7) from the incubator. Remove the culture medium and wash the cells with prewarmed PBS.
2. Add 10 mL of DMEM high glucose medium supplemented with 10% FBS, 50 µg/mL ascorbic acid, and 1% pen/strep, prewarmed at 37 °C.
3. Place the Petri dish at 37 °C in a 5% CO₂ humidified atmosphere. Change the medium every 2 days.
4. Monitor the NHDFs under a microscope for 14 days.

4. 3D maturation

1. Take out the 10 cm cell culture dish from the incubator and remove the DMEM high glucose medium.
2. Gently and quickly detach the formed cell sheet from the dish with a cell scraper. While detaching, simultaneously roll the cell sheet into a 3D rod-like organoid.
3. Pick up and place the NHDF organoids in a 10 cm non-adherent (for cells) Petri dish. This dish type is used to avoid cell outmigration from the organoid to the plastic.
4. At this time point or a day after (day 0 or day 1 of 3D maturation), collect some of the organoids for further analyses such as wet weight, histological evaluation (day 1 organoids are preferable as they become more compact), RNA and protein isolation.
5. Fix the edges of the organoid with metal pins as follows:
   1. Hold one side of the organoid carefully with a tweezer, and with another tweezer, gently apply manual stretching of approximately 10% axial elongation. To estimate the 10% axial elongation, use a millimeter paper or ruler.
   2. Next, pick up one metal pin with the tweezer and manually press down through the organoid edge into the plastic dish to fix it. Repeat this procedure with the second pin onto the second edge of the organoid.
6. Add 10 mL of DMEM high glucose medium supplemented with 10% FBS, 1x MEM amino acids, 50 µg/mL ascorbic acid, 10 ng/mL TGFß3, and 1% pen/strep, prewarmed at 37 °C.
7. Place the 10 cm dish at 37 °C in a 5% CO₂ humidified atmosphere. Change the medium every 2 days.
8. Monitor the organoids regularly for 14 days.
   NOTE: Pins may loosen up, hence re-refix using the same technique as described in step 4.5.
9. After 14 days, remove the DMEM high glucose medium from the organoids. Wash the organoids with prewarmed PBS.
10. Measure organoids for wet weight at day 0 and day 14 using a precision scale.
    1. Place an empty 10 cm dish on the scale and tare the weight to zero to ensure that only the weight of the organoids is measured. Remove the culture medium fully from the 10 cm dish and measure the organoid wet weight one by one.
       NOTE: In this study, at day 0, three organoids per donor, and at day 14, two organoids per donor were weighed.
11. According to the study purposes, implement some of the NHDF organoids in further histological analyses or immediately freeze them in liquid nitrogen using sterile DNA/RNA-free tubes for subsequent DNA, RNA, and protein isolation and then store them at -80 °C.
12. For histological investigation, fix each organoid in 5 mL of precooled (4 °C) 4% paraformaldehyde in PBS (adjusted to neutral pH) for 45 min on ice, followed by PBS washing at RT (2 x 5 min each).
13. Cryoprotect the fixed organoids using a sucrose gradient by placing the organoids in glass jars for histology. Incubate the organoids in 10% sucrose in PBS solution for 2 h at 4 °C, following 20% sucrose/PBS for 2 h at 4 °C, and finally, 30% sucrose/PBS overnight incubation at 4 °C. Change the sucrose solutions by first pipetting out and then filling up with the new solution.
14. Embed the organoids in cryo-medium.
    1. Place a copper plate onto dry ice in a Styrofoam box and cool down for 10 min.
    2. Next, place a plastic cryomold, prefilled with cryo-medium, on the copper plate and gently press the organoid to the bottom of the cryomold using tweezers. Wait until the cryomedium is fully frozen.
    3. For long organoids, first cut in half with a scalpel and put the two halves parallel to each other in the cryomold. Repeat the procedure for each organoid intended to undergo histological analysis.
15. Store the samples at -20 °C till cryosectioning.
16. Using a cryotome, cut the organoids longitudinally in 10 µm thick sections and subject to histological staining such as hematoxylin and eosin (H&E) using standard protocol⁸.

Results

The 3D T/L organoid model was previously established and demonstrated here by implementing commercially purchased NHDF (n=3, 3 organoids per donor, NHDF were used at passages 5-8). The model workflow is summarized in Figure 1. Figure 2 shows representative phase-contrast images of NHDF culture during the pre-expansion in T-75 flasks (Figure 2A) as well as at the beginning and after 5 days of culture in the 2D expansion step in 10 cm...

Discussion

The results demonstrated in this study provide valuable insights into the establishment and characterization of the NHDF 3D organoid model for studying T/L tissues. The 3-step protocol led to the formation of 3D rod-like organoids that exhibit typical features of T/L niche. This model was previously reported in Kroner-Weigl et al. 2023⁷ and demonstrated in great detail here.

The phase-contrast images presented in Figure 2 showed th...

Materials

Name	Company	Catalog Number	Comments
Ascorbic acid	Sigma-Aldrich, Taufkirchen,Germany	A8960
10 cm adherent cell culture dish	Sigma-Aldrich, Taufkirchen,Germany	CLS430167
10 cm non-adherent petri dish	Sigma-Aldrich, Taufkirchen,Germany	CLS430591
Cryo-medium	Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands	4583
Cryomold standard	Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands	4557
D(+)-Sucrose	AppliChem Avantor VWR International GmbH, Darmstadt, Germany	A2211
DMEM high glucose medium	Capricorn Scientific, Ebsdorfergrund, Germany	DMEM-HA
DMEM low glucose	Capricorn Scientific, Ebsdorfergrund, Germany	DMEM-LPXA
Fetal bovine serum	Anprotec, Bruckberg, Germany	AC-SM-0027
Fibroblast growth medium 2	PromoCell, Heidelberg, Germany	C-23020
Inverted microscope with high resolution camera	Zeiss	NA	Zeiss Axio Observer with  Axiocam 506
MEM amino acids	Capricorn Scientific, Ebsdorfergrund, Germany	NEAA-B
Metal pins	EntoSphinx, Pardubice, Czech Republic	04.31
Normal human dermal fibroblasts	PromoCell, Heidelberg, Germany	C-12302
Paraformaldehyde	AppliChem, Sigma-Aldrich, Taufkirchen, Germany	A3813
Penicillin/streptomycin	Gibco, Thermo Fisher Scientific, Darmstadt, Germany	15140122
Phosphate buffer saline	Sigma-Aldrich, Taufkirchen, Germany	P4417
TGFß3	R&D Systems, Wiesbaden, Germany	8420-B3
Trypsin-EDTA 0,05% DPBS	Capricorn Scientific, Ebsdorfergrund, Germany	TRY-1B