D.D. and S.M.-D. acknowledge the BMBF Grant &#34;CellWiTaL: Reproducible cell systems for drug research - transfer layer-free laser printing of highly specific single cells in three-dimensional cellular structures&#34; Proposal Nr. 13N15874. D.D. and V.R.A. acknowledge the EU MSCA-COFUND Grant OSTASKILLS &#34;Holistic training of next-generation Osteoarthritis researches&#34; GA Nr. 101034412. All authors acknowledge Mrs. Beate Geyer for technical assistance.

NOTE: All the steps must be conducted using aseptic techniques.
1. Culture and pre-expansion of NHDFs
<ol>
	<li>Rapidly thaw the cryo-vial containing adult cryopreserved normal human dermal fibroblasts (NHDFs, 1 x 106 cells) at 37 &#176;C until they are almost defrosting.</li>
	<li>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 &#176;C to the cells.</li>
	<li>Transfer the cells to a 15 mL centrifuge tube. Centrifuge the cells at 300 x g for 5 min at room temperature (RT).</li>
	<li>Remove the supernatant. Resuspend the cell pellet in 12 mL of prewarmed NHDF medium.</li>
	<li>Transfer the cells to a T-75 flask and shake briefly in a cross-wise manner. Place the T-75 flask at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Change the medium every 2 days. Observe the NHDFs under a microscope until they reach 70% - 80% confluency.</li>
</ol>
2. 2D expansion
<ol>
	<li>Take out the T-75 flask of the incubator and remove the NHDF medium. Wash the cells with prewarmed phosphate buffer saline (PBS).</li>
	<li>Add 3 mL of 0.05% trypsin-EDTA to the cells. Incubate the cells at 37 &#176;C in a 5% CO2 humidified incubator until the cells detach from the flask (approximately 3 min).</li>
	<li>Add 6 mL of prewarmed NHDF medium to neutralize the trypsin action. Transfer the cells to a 15 mL centrifuge tube.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at RT and remove the supernatant.</li>
	<li>Resuspend the cell pellet in Dulbecco&#39;s Modified Eagles Medium (DMEM) low glucose supplemented with 10% fetal bovine serum (FBS), 1x MEM amino acids, 1% pen/strep, prewarmed at 37 &#176;C.</li>
	<li>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.</li>
	<li>Place the 10 cm cell culture dish at 37 &#176;C in a 5% CO2 humidified incubator. Change the medium every 2 days</li>
	<li>Monitor the cells under a microscope until reaching 100% confluency (ca. 5 days).</li>
</ol>
3. 2D stimulation
<ol>
	<li>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.</li>
	<li>Add 10 mL of DMEM high glucose medium supplemented with 10% FBS, 50 &#181;g/mL ascorbic acid, and 1% pen/strep, prewarmed at 37 &#176;C.</li>
	<li>Place the Petri dish at 37 &#176;C in a 5% CO2 humidified atmosphere. Change the medium every 2 days.</li>
	<li>Monitor the NHDFs under a microscope for 14 days.</li>
</ol>
4. 3D maturation
<ol>
	<li>Take out the 10 cm cell culture dish from the incubator and remove the DMEM high glucose medium.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;1&#160;organoids are preferable as they become more compact), RNA and protein isolation.</li>
	<li>Fix the edges of the organoid with metal pins as follows:
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Add 10 mL of DMEM high glucose medium supplemented with 10% FBS, 1x MEM amino acids, 50 &#181;g/mL ascorbic acid, 10 ng/mL TGF&#223;3, and 1% pen/strep, prewarmed at 37 &#176;C.</li>
	<li>Place the 10 cm dish at 37 &#176;C in a 5% CO2 humidified atmosphere. Change the medium every 2 days.</li>
	<li>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.</li>
	<li>After 14 days, remove the DMEM high glucose medium from the organoids. Wash the organoids with prewarmed PBS.</li>
	<li>Measure organoids for wet weight at day 0 and day 14 using a precision scale.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C.</li>
	<li>For histological investigation, fix each organoid in 5 mL of precooled (4 &#176;C) 4% paraformaldehyde in PBS (adjusted to neutral pH) for 45 min on ice, followed by PBS washing at RT (2 x&#160;5 min each).</li>
	<li>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 &#176;C, following 20% sucrose/PBS for 2 h at 4 &#176;C, and finally, 30% sucrose/PBS overnight incubation at 4 &#176;C. Change the sucrose solutions by first pipetting out and then filling up with the new solution.</li>
	<li>Embed the organoids in cryo-medium.
	<ol>
		<li>Place a copper plate onto dry ice in a Styrofoam box and cool down for 10 min.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Store the samples at -20 &#176;C till cryosectioning.</li>
	<li>Using a cryotome, cut the organoids longitudinally in 10 &#181;m thick sections and subject to histological staining such as hematoxylin and eosin (H&#38;E) using standard protocol8.</li>
</ol>

Three-Step Organoid Model for Tendon Research and Engineering

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The authors have no conflicts of interest to declare.

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 the progression of the NHDF confluence and sheet formation during the expansion and stimulation steps. The 3D maturation process was performed by manually rolling the cell sheets into 3D rod-like organoids, which were then cultured under static tension for 14 days. The organoids displayed a glistening white appearance and increased density by day 14, suggesting contraction and structural reorganization within the organoids. This indicated that the maturation period is critical for achieving a more organized and T/L tissue-mimicking structure.
Furthermore, weight analysis revealed a significant decrease in the wet weight of organoids between days 0 and 14, yet again indicating significant contraction and ECM reorganization within the organoids during the maturation process. Importantly, wet weight measurements may vary between experiments due to handling and residual medium within the organoid or in the dishes. Moreover, the organoid might contain more medium at the beginning because, as discussed below, at day 0, the layers formed by the cell sheet rolling are segregated and not fused. With time, the layers joined and became more compact, and thus, the medium might be extruded.
Morphological evaluation by H&#38;E staining provided additional insights into the organoid structural characteristics. On day 1, the organoids exhibited disconnected layers, primarily composed of cells with round nuclei surrounded by thin peri-cellular ECM. The lack of cell alignment parallel to the axis of the tensile load suggested that further maturation is needed to achieve a more organized cellular and ECM architecture of the organoids. Despite the contraction and organoid weight and dimension reduction between days 1 and 14, the H&#38;E staining showed an increase in eosin-positive areas, suggesting augmented ECM deposition. Moreover, there were no longer visible layers, and occasionally, the cells had elongated nuclei and were positioned in parallel cell rows. This pointed out that the cells within the organoids undergo self-organization by changing their own arrangement as well as ECM deposition, structure, and alignment, thus mimicking the behavior of the native T/L tissue formation.
Overall, these findings highlight the potential of the NHDF organoid model as a valuable tool for studying T/L biology, in vitro tenogenesis, and even for the further development of scaffold-free T/L engineering. However, there are three critical steps for the successful handling of the 3D organoid model: 1) The formation of a tight cell sheet during 2D stimulation stage of the protocol. When using primary cells, this predominantly depends on the donor cell properties; therefore, it is important to assess multiple donors in parallel; 2) The manual rolling of the cell sheet. This requires rolling quickly, but at the same time gently, the cell sheet using a cell scraper. Therefore, it is advisable to plan initial organoids to first train this procedure; and 3) The stable fixation of the pins at the organoid edges. This fixation ensures the organoid axial elongation and prevents deformities as well as collapsing into lump-like structure. Moreover, during the 3D maturation stage, since the organoids experience remodeling of the ECM, the pins can suddenly detach, therefore, it is important to frequently monitor each organoid and to re-fix the pins.
The current model has not yet reached the level of in vivo mimicry to T/L tissue and harbors a number of limitations. For example, it requires a large number of cells per organoid as well as 35 days for the procedure to be completed, while most differentiation protocols, such as 3D chondrogenic pellet model, are set to 21 days22. The washing steps in the protocol procedure should preferably be carried out with physiologically-balanced wash buffer, for example, Hanks Balanced Salt Solution (HBSS) or Earles Balanced Salt Solution (EBSS), rather than PBS which might have metabolic consequences for primary human cells cultivated in vitro. Regarding the culture medium used in the 2D stimulation and 3D maturation stages in the organoid, it has been chosen DMEM plus 10% FBS formulation because such is the foundation of widely accepted differentiation protocols as well as with the intention to standardize and compare between different cell types for their organoidogenic properties23. Reproduction of the 3-step organoid protocol across different laboratory settings might be influenced by cell type used, cell quality and passage, as well as donor-dependent cell properties. Variations in the equipment, especially in the quality of the 10 cm cell culture dishes24 used during the 2D stimulation stage, could potentially impact the robustness of the cell sheet formation. Moreover, due to ECM contraction over the 3D maturation, the axial fixation of the organoids via pins might loosen up; therefore, as mentioned above, frequent monitoring is recommended. Testing and selecting different pins in terms of diameter and strength can also reduce the risk of pin detachment25. However, using pins for manual stretching is not robust, and it is prone to handling variability; hence, it is very important to develop in follow-up research a clamping mechanism for holding the organoid edges, which can result not only in standardization of this step, but also can permit dynamic stretching of the organoids. In general, it will be of interest to investigate two ways of organoid model modification, one downscaling - to reduce the number of cells per organoid and procedure time, thus making it more user-friendly, especially for in vitro studies; and the second upscaling - to increase the dimensions of the organoids in order to test their behavior in large animal models as potential tendon replacement strategy.
The model demonstrated here can provide a platform for investigating the mechanisms underlying tendon development, pathophysiology, and perhaps repair and regeneration if the organoids are combined with different cell types and subjected to micro-ruptures. It is important to mention that the study of Kroner-Weigl et al. 20237 identified several limitations regarding the NHDF cell type, namely, the NHDFs formed organoids are larger and more cellular than those derived from T/L cells, making it challenging to control cell proliferation and behavior. With regards to cell apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-based assay showed no evidence of dead cells throughout the organoid at day 147, suggesting appropriate diffusion of nutrients, waste, and oxygen during the 3D maturation step. Interestingly, despite the morphological differences, pilot screening by quantitative PCR of a number of T/L-related genes (including different collagen types, Scleraxis (SCX), Mohawk (MKX), etc.) suggested comparable gene expression between NHDF and T/L organoids7, a finding that should be further investigated as well as should be validated at the protein level. Hence, for this cell type, further optimization is required in order to support T/L differentiation and can be based on adjusting the medium used in 2D stimulation and 3D maturation steps (e.g., serum concentration, growth factors, vitamins), extending the time of maturation or even subjecting the organoids to dynamic mechanical stretch. Evolving the protocol could further improve the organoid compositional, structural, and functional properties, thus enabling the model to better mimic the complex in vivo T/L tissue niche. Moreover, alternative cell sources can be investigated for their capacity to form T/L organoids and whether they can undergo T/L differentiation. Yan et al. 20209, employed the 3D organoid model by using young/healthy and aged/degenerative tendon stem/progenitor cells (Y-TSPCs and A-TSPCs) to investigate cellular behavior in 3D as well as whether tendon-forming capacity changes with T/L aging. They reported that A-TSPC 3D organoids exhibit poor tissue morphology, and the cells within have a lower proliferation rate but higher apoptosis paralleled with elevated levels of senescence-related markers9. Hence, the T/L 3D organoid model is a promising platform for studying molecular and cellular mechanisms involved in tendon aging and further can be used to test novel pharmacological therapies for tendon repair and regeneration. In a different approach, Chu et al. 2021 implemented dental follicle cells (DFCs) in the 3D organoid model in order to assess their ligamentogenic differentiation. This cell type formed very well-organized organoids, thus suggesting DFCs as an attractive cell source to differentiate in periodontal ligament (PDL) tissue, which could, in turn, develop a promising therapeutic strategy for periodontitis8.
In the future, the model could become even more complex by including different cell types (e.g., myofibroblasts, macrophages), which may offer novel insights into cell-specific behaviors and cellular crosstalk within organoids and contribute to a more comprehensive understanding of T/L biology, physiology and pathophysiology8,9,16,26.
The 3D organoid models hold several advantages for tissue engineering and regenerative medicine applications. They mimic closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of human tissues, thereby offering a physiologically relevant platform for studying disease mechanisms and drug responses27. Organoids can be used as a high-throughput screening platform to assess the effectiveness but also potential side effects of different drugs and provide a reliable and low-cost alternative to animal testing. Furthermore, they enable direct comparisons between healthy and diseased conditions, allowing the discovery of key molecular and cellular changes associated with diverse pathologies, identification of biomarkers for early disease detection as well as facilitation of ascended understanding of disease mechanisms, and the development of targeted therapies28,29.
Taken together, we demonstrated in great detail the steps of the previously established 3D T/L organoid model that provides a promising platform for studying T/L tissue biology and in vitro tenogenic processes of various cell types. Further implementation, optimization, and research of this model are strongly encouraged as they may lead to advancing scaffold-free T/L engineering strategies, which, in turn, can contribute to improving T/L therapy.

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.

<table><tbody><tr><td>Ascorbic acid&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich, Taufkirchen,Germany&amp;nbsp;&amp;nbsp;</td><td>A8960</td><td></td></tr><tr><td>10 cm adherent cell culture dish</td><td>Sigma-Aldrich, Taufkirchen,Germany&amp;nbsp;</td><td>CLS430167</td><td></td></tr><tr><td>10 cm non-adherent petri dish&amp;nbsp;</td><td>Sigma-Aldrich, Taufkirchen,Germany&amp;nbsp;</td><td>CLS430591</td><td></td></tr><tr><td>Cryo-medium</td><td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&amp;nbsp;&amp;nbsp;</td><td>4583</td><td></td></tr><tr><td>Cryomold standard&amp;nbsp;</td><td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands</td><td>4557</td><td></td></tr><tr><td>D(+)-Sucrose&amp;nbsp;</td><td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td><td>A2211</td><td></td></tr><tr><td>DMEM high glucose medium&amp;nbsp;</td><td>Capricorn Scientific, Ebsdorfergrund, Germany&amp;nbsp;</td><td>DMEM-HA</td><td></td></tr><tr><td>DMEM low glucose</td><td>Capricorn Scientific, Ebsdorfergrund, Germany&amp;nbsp;&amp;nbsp;</td><td>DMEM-LPXA</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Anprotec, Bruckberg, Germany&amp;nbsp;</td><td>AC-SM-0027</td><td></td></tr><tr><td>Fibroblast growth medium 2&amp;nbsp;</td><td>PromoCell, Heidelberg, Germany&amp;nbsp;&amp;nbsp;</td><td>C-23020</td><td></td></tr><tr><td>H&amp;amp;E staining kit</td><td>Abcam</td><td>ab245880</td><td></td></tr><tr><td>Inverted microscope with high resolution camera</td><td>Zeiss</td><td>NA</td><td>Zeiss Axio Observer with&amp;nbsp; Axiocam 506</td></tr><tr><td>MEM amino acids&amp;nbsp;</td><td>Capricorn Scientific, Ebsdorfergrund, Germany&amp;nbsp;&amp;nbsp;</td><td>NEAA-B</td><td></td></tr><tr><td>Metal pins&amp;nbsp;</td><td>EntoSphinx, Pardubice, Czech Republic&amp;nbsp;</td><td>04.31</td><td></td></tr><tr><td>Normal human dermal fibroblasts&amp;nbsp;</td><td>PromoCell, Heidelberg, Germany&amp;nbsp;</td><td>C-12302</td><td></td></tr><tr><td>Paraformaldehyde</td><td>AppliChem, Sigma-Aldrich, Taufkirchen, Germany&amp;nbsp;</td><td>A3813</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffer saline&amp;nbsp;</td><td>Sigma-Aldrich, Taufkirchen, Germany&amp;nbsp;</td><td>P4417</td><td></td></tr><tr><td>TGF&amp;szlig;3&amp;nbsp;</td><td>R&amp;amp;D Systems, Wiesbaden, Germany&amp;nbsp;</td><td>&amp;nbsp;8420-B3</td><td></td></tr><tr><td>Trypsin-EDTA 0,05% DPBS&amp;nbsp;</td><td>Capricorn Scientific, Ebsdorfergrund, Germany&amp;nbsp;</td><td>&amp;nbsp;TRY-1B</td><td></td></tr></tbody></table>

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.

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 cell culture dishes (Figure 2B). Figure 2C (representative phase-contrast images) demonstrates the confluence of NHDFs during 2D stimulation after 14 days of culture in the 10 cm cell culture dishes. Subsequently, the cell sheets were manually detached and simultaneously rolled into 3D rod-like organoids (at day 0, organoid length is approximately 6 cm, width approximately 0.4 cm), which were cultivated under 10 % static tension for 14 days (Figure 3, day 0 and day 14, representative macroscopic images). By day 14 of the 3D maturation process, the organoids displayed a glistening white appearance, contracted, and appeared denser than the initial organoids. The wet weight of the organoids was measured at the time of formation (day 0) and again on day 14 of the 3D maturation step (Figure 4). A significant reduction in the wet weight was observed, indicating contraction and ECM structural reorganization within the organoids during the time span of this protocol step. In addition to weight change, due to the contraction, the organoid length also reduced (at day 14, the organoid length is approximately 3.5 cm, and the width is approximately 0.3 cm). In this process, the metal pins loosened up and had to be re-fixed; hence, during the 14 days of 3D maturation, we recommend monitoring the organoids frequently. To evaluate the morphology of the organoids, longitudinal sections were obtained from NHDF organoids collected on day 1 and day 14 of the 3D maturation step and subjected to H&#38;E staining (Figure 5). On day 1, the organoids displayed disconnected layers, primarily composed of cells surrounded by low amounts of ECM. Furthermore, the cells within the organoid layers were not aligned along the longitudinal organoid axis used as direction for the applied static tensile load. The NHDF nuclei exhibited a round shape with varying sizes and shapes. H&#38;E analysis of day 14 organoids revealed a noticeable increase in the eosin signal, indicating ECM deposition during the 3D maturation process. At this time point, the organoids exhibited fused layers, with areas containing aligned cells. Cells were frequently in groups; however, they were also organized in cell rows in some locations. The majority of the nuclei had similar sizes and occasionally were elongated.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66047/66047fig1v2.jpg" /> 
Figure 1: Cartoon of 3D T/L organoid model composed of 3 steps: 2D expansion, 2D stimulation, and 3D maturation. Initially, NHDFs were pre-expanded in a T-75 flask until they reached 70%-80% confluence. Subsequently, NHDFs were cultivated using Dulbecco&#39;s Modified Eagles Medium (DMEM) low glucose medium in a 10 cm cell culture dish for 5 days (2D expansion). The cells were then stimulated with DMEM high glucose medium supplemented with ascorbic acid for 14 days of culture (2D stimulation). Following, NHDF were detached from the 10 cm adherent dish, rolled into a 3D rod-like organoid, transferred, and fixed in a 10 cm non-adherent cell culture dish filled with DMEM high glucose medium supplemented with ascorbic acid and Transforming Growth Factor beta 3 (TGF&#223;3) for 14 days (3D maturation). The formed 3D organoids may undergo an assessment of wet weight, histological evaluation, RNA and protein isolation, and other analyses (e.g., transmission electron microscopy, biomechanical measurements) at different time points such as days 0, 1, and 14. The cartoon was generated in BioRender software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66047/66047fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66047/66047fig02.jpg" /> 
Figure 2: Representative phase-contrast images of NHDFs during pre-expansion, 2D expansion, and 2D stimulation steps. (A)&#160;NHDFs during pre-expansion in T-75 flask. (B) NHDFs at days 1 and 5 of the 2D expansion step in a 10 cm cell culture dish. (C) NHDFs at days 1 and 14 of 2D stimulation step in a 10 cm cell culture dish. Scale bars: 200 &#181;m. Images were taken with an inverted microscope equipped with a high-resolution camera. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66047/66047fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66047/66047fig03.jpg" /> 
Figure 3: Gross morphology of NHDF 3D organoids. Representative macroscopic images of NHDF organoids at days 0 and 14 of the 3D maturation step. Scale bar: 1 cm. Images were taken with a mobile phone camera. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66047/66047fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66047/66047fig04.jpg" /> 
Figure 4: Wet weight analysis of NHDF 3D organoids. Organoids&#39; wet weight measurement was at day 0 and day 14, the end of the 3D maturation process. The graph shows mean values and standard deviations (NHDF n = 3, 3 organoids/donor at day 0 and 2 organoids/donor at day 14 due to 1 organoid/donor taken for histology at day 1), each dot represents individual organoids while each colored shape represents a different individual donor. Data is presented as mean &#177; standard deviation, and an unpaired parametric t-test was used. Statistical differences: **p &#8804;0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66047/66047fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66047/66047fig05.jpg" /> 
Figure 5: Histological analysis of NHDF 3D organoids. Representative images of hematoxylin and eosin (H&#38;E) stained sections of NHDF organoids during the 3D maturation process at days 1 and 14. Right image - low magnification images, left image - magnified view with indicators as follows: yellow arrows - disconnected layers; yellow arrowheads - nuclei with varying sizes and shapes; black arrows - fused layers and ECM deposition; black arrowheads - cell rows, nuclei with similar size and elongation; black stars -cells in groups. Scale bars: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66047/66047fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

demonstration-self-assembled-cell-sheet-culture-manual-generation-3d

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.2K Views.  University of Würzburg. We demonstreren een eerder opgesteld en implementeren organoïdeprotocol in drie stappen met behulp van in de handel verkrijgbare, normale menselijke dermale fibroblasten als celbron. Het 3D-organoïdemodel heeft verschillende voordelen voor weefselmanipulatie en toepassing in regeneratieve geneeskunde. Het bootst nauwkeurig de cellulaire samenstelling en organisatie na, evenals cel-tot-cel en cel-tot-matrix interacties van peesweefsels.De gevestigde 3D-pees-ligament-organoïdebenaderi...

Video: Auteur in de schijnwerpers: Geavanceerde peesweefselmanipulatie met 3D-organoïde modellen

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Developmental Biology

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

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.

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.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

Medicine

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

Organotypic 3D Skin Culture: A Cassette-based In Vitro Culture Technique to Regenerate Human Epidermis on Acellular Dermis

Source: Jingting Li et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/53280"> Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis</a>, J. Vis. Exp. (2015).
This video protocol describes a cassette-based in vitro culture technique to generate&#160;human organotypic skin using epidermal keratinocytes and devitalized human dermis. They mimic the function and physiology of their in vivo tissue counterparts.

Source: Jingting Li et al.  Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis, J. Vis. Exp. (2015).
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Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

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