This study was sponsored by National Natural Science Foundation of China (NSFC 81700978), Fundamental Research Funds for the Central Universities (1504219050), Natural Science Foundation of Shanghai (17ZR1432800), and Shanghai Medical Exploration Project (17411972600).

The study protocol was approved by the Ethics Committee of School and Hospital of Stomatology, Tongji University. All patients provided written informed consent.
1. PDL cell isolation
<ol>
	<li>Make proliferation medium for culture of PDL cells: &#945;-MEM medium supplemented with 10% FCS and 100 U/mL pen/strep.</li>
	<li>Prepare a container with ice to transfer isolated third molars.</li>
	<li>Sterilize surgical instruments by using an autoclave.</li>
	<li>Extract normal human impacted third molars from adults (18-28 years of age) at the Dental Clinic of School and Hospital of Stomatology, Tongji University.</li>
	<li>Place third molars into the proliferation medium and transfer into the laboratory at 4 &#176;C.</li>
	<li>Place the extracted tooth in a sterile 100 mm Petri dish, working within a biohazard laminar flow hood.</li>
	<li>Add 10 mL of PBS to wash the extracted tooth. Repeat the washing step twice.</li>
	<li>By using a sterile scalpel, gently separate the PDL from the root. 
	NOTE: PDL should be scraped from the middle third part of the root to avoid contaminations from gingival or apical tissues.</li>
	<li>Mince PDL into 1-2-mm fragments with the help of a sterilized scalpel blade.</li>
	<li>Place PDL fragments in a T-25 culture flask, filled with 3-4 mL of proliferation medium.</li>
	<li>Culture the samples in an incubator at 37 &#176;C in 5% CO2 with humidified air.</li>
	<li>Change the culture medium after outgrowth of PDL cells was observed. It usually takes 1-2 weeks for the outgrowth of PDL cells. Observe the outgrowth of PDL cells via an inverse phase contrast microscope.</li>
	<li>Change the culture medium twice per week thereafter.</li>
	<li>When PDL cells have reached confluence, sub-culture cells at ratio of 1:4 (passage 1).</li>
	<li>Observe cell growth daily and change the culture medium twice per week.</li>
	<li>Perform each subsequent passage at ratio of 1:4 after the cells achieve 80% confluence. Use third- to fifth-generation of PDL cells for cell seeding.</li>
</ol>
2. Preparation of chitosan films
<ol>
	<li>For the preparation of a 1% (v/v) acetic acid solution, add 5 mL of acetic acid to 495 mL of double-distilled water in a clean glass beaker.</li>
	<li>For the preparation of 1% w/v chitosan solution, weigh 5 g of chitosan powder (average molecular weight 400 kDa, 85% degree of acetylation) and add to 495 mL of 1% (v/v) acetic acid solution.</li>
	<li>Stir the prepared solution for 24 hours with stirring bar and magnetic stirring plate at 60 &#176;C.</li>
	<li>Autoclave the prepared solution to ensure complete dissolution of chitosan. 
	NOTE: The experiment can be paused at this stage.</li>
	<li>To form chitosan films, add 1% w/v chitosan solution into tissue culture polystyrene (TCPS) dishes at 0.25 mL/cm2. For example, add 0.5 mL of 1% w/v chitosan solution to one well of 24 &#8211;well plate.</li>
	<li>Dry TCPS dishes in an oven at 60 &#176;C for 24 hours to form chitosan films.</li>
	<li>Prepare 0.5 N sodium hydroxide (NaOH). Stir 10 g of NaOH, a little at a time, into a large volume of double-distilled water by using the magnetic stir bar, and then dilute the solution to make 0.5 L. 
	NOTE: Add NaOH to water--do not add water to solid NaOH. Do not touch NaOH! It could cause chemical burns.</li>
	<li>Neutralize chitosan films with 0.5 N NaOH. Add 0.5 mL of 0.5 N NaOH solution to one well of 24-well plate for 2 hours.</li>
	<li>Wash chitosan films three times with double-distilled water. 
	NOTE: The experiment can be paused at this stage.</li>
	<li>Prior to cell seeding, sterilize the chitosan-coated 24-well plates in 70% alcohol overnight at room temperature.</li>
	<li>Rinse chitosan-coated plates three times with PBS at room temperature.</li>
	<li>Sterilize chitosan-coated plates by treatment under ultraviolet light overnight at room temperature.</li>
</ol>
3. Cell seeding
<ol>
	<li>Pre-warm proliferation medium PBS solution and trypsin/EDTA solution to 37 &#176;C.</li>
	<li>Discard the supernatant from the T-75 cell culture flask.</li>
	<li>Wash confluent PDL cells with 10 mL of PBS solution.</li>
	<li>Discard the PBS solution.</li>
	<li>Add 1 mL of trypsin/EDTA solution to T-75 cell culture flask.</li>
	<li>Incubate cells with trypsin/EDTA solution for 3 min at 37 &#176;C.</li>
	<li>Terminate the digestion process of trypsin by adding 3 mL of proliferation medium to T-75 cell culture flask.</li>
	<li>Transfer the prepared cell suspension into a 15 mL conical centrifuge tube.</li>
	<li>Centrifuge the suspension for 5 min at 300 x g and room temperature.</li>
	<li>Discard the supernatant from the 15 mL conical centrifuge tube and re-suspend the cell pellet in 500 &#181;L of proliferation medium.</li>
	<li>Count PDL cells with a hemocytometer.</li>
	<li>Seed PDL cells at the densities of 0.5 x 104, 1 x 104, 3 x 104, and 6 x 104 cells/cm2.</li>
	<li>Culture PDL cells on chitosan films in an incubator at 37 &#176;C in 5% CO2 with humidified air.</li>
	<li>Change the culture medium twice per week thereafter.</li>
	<li>On days 1, 2, 3, 4, and 5, observe the cell morphology daily via an inverse phase contrast microscope.</li>
</ol>
4. Cell survival
<ol>
	<li>After 1, 3, and 6 days of culture, determine the survival of cells using a commercial viability/cytotoxicity kit.</li>
	<li>In a 15 mL tube, prepare 2 mL of a fresh solution of 2 mM calcein-AM and 4 mM ethidium homodimer in PBS by vortexing for 5 s. 
	NOTE: Keep the process in the dark.</li>
	<li>Remove the culture medium and wash spheroids in PBS.</li>
	<li>Incubate spheroids in of PBS containing 2 mM calcein-AM and 4 mM ethidium homodimer for 30 min at room temperature in the dark.</li>
	<li>Rinse spheroids twice in PBS. For spheroids from one well of 24-well plate, use 2 mL of PBS for each time.</li>
	<li>Observe the samples by using a fluorescence microscope using 10x or 20x magnification.</li>
</ol>

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M., Jin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+tissue+engineering+and+regeneration:+current+approaches+and+expanding+opportunities.">Periodontal tissue engineering and regeneration: current approaches and expanding opportunities.</a> Tissue Engineering Part B Review. 16 (2), 219-255 (2010).</li><li>Yu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+periodontal+tissue+regeneration+by+periodontal+cell+implantation.">Enhanced periodontal tissue regeneration by periodontal cell implantation.</a> Journal of Clinical Periodontology. 40 (7), 698-706 (2013).</li><li>Yan, X. Z., Yang, F., Jansen, J. A., de Vries, R. B., van den Beucken, J. 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The authors have nothing to disclose.

The present study introduced a 3D cell culture system to overcome some limitations related to conventional 2D monolayer cell culture. According to the protocol, PDL cellular spheroids were successfully formed by culturing cells on chitosan films. Our previous study reported that spheroid formation increased the self-renewal and osteogenic differentiation capacities of PDL cells14. Instead of using an enzyme to harvest cells from TCPS, PDL cell spheroids could be harvested from chitosan films by si...

Periodontitis, initialized principally by dental plaque1, is characterized by the damage of periodontal tissues including periodontal ligament (PDL), alveolar bone, and cementum. Current treatments for periodontitis are usually successful in preventing the progress of the active disease, but the regeneration of lost periodontal tissues remains a clinical challenge. Recently, important progress has been made in cell-based approaches for periodontal tissue regeneration to overcome the drawbacks of current treatments2,3,4.
Our previous systematic review revealed that PDL cells showed great potential for periodontal regeneration5. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture6. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment7. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells8. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture.
Conventional 3D culture method is encapsulating cells in 3D biomaterials. Compared with cells encapsulated in 3D biomaterials, cellular spheroids mimic the in vivo situation more closely because spheroids are aggregates of cells growing free of foreign materials9,10,11,12. It is reported that cellular spheroids promoted MSC bioactivities via the preservation of extracellular matrix (ECM) components including fibronectin and laminin13. To improve conventional PDL cell culture models, we have recently developed a 3D PDL cell culture method, which is based on spheroid formation of PDL cells on chitosan films14. Spheroid formation increased the self-renewal and osteogenic differentiation capacities of PDL cells14. Here, we present detailed PDL cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the shortcomings related to conventional TCPS cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

<table><tbody><tr><td>&amp;alpha;-MEM</td><td>Gibco</td><td>11900-073</td><td></td></tr><tr><td>acetic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>64197</td><td></td></tr><tr><td>Cell culture flask 25 cm&lt;sup&gt;2</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Cell culture flask 75 cm&lt;sup&gt;2</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>Chitosan</td><td>Heppe Medical&amp;nbsp;Chitosan GmbH</td><td>/</td><td>molecular weight 500 kDa, degree of deacetylation 85%</td></tr><tr><td>FCS</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Molecular&amp;nbsp;Probes</td><td>L3224</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>1310732</td><td></td></tr><tr><td>PBS</td><td>KeyGen Biotech&amp;nbsp;</td><td>KGB5001</td><td></td></tr><tr><td>pen/strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Trypsin/EDTA&amp;nbsp;</td><td>KeyGen Biotech&amp;nbsp;</td><td>KGM25200</td><td></td></tr><tr><td>15 mL conical centrifuge tube</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3524</td><td></td></tr></tbody></table>

formation of human periodontal ligament cell spheroids on chitosan films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Using the present protocol, viable PDL cell spheroids were successfully formed. Figure 1 showed that suspended cells or spheroids instead of attached cells were mainly observed on chitosan films. For the seeding density of 0.5 x 104 cells/cm2, attached PDL cells were occasionally found on day 1 and 3, and PDL cell spheroids were rarely observed. On the contrary, for the seeding densities of 3 x 104 and 6 x 104 cells...

Watch this Scientific Journal Video about Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films at JoVE.com

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

formation-human-periodontal-ligament-cell-spheroids-on-chitosan

Research

JoVE Journal

Developmental Biology

5.3K Views.  Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration. Dit is een gedetailleerd protocol van het kweken van parodontale ligament cel sferoïden door chitosan films. Vergeleken met het conventionele kweeksysteem produceert het sferoïde kweeksysteem parodontale ligamentcellen met verhoogde zelfvernieuwing en osteogene differentiatievaardigheden. Om te beginnen, bereid 1%gewicht-tot-volume chitosan oplossing zoals aangegeven in het manuscript.Voeg 0,5 milliliter chitosan oplossing in elke put van een 24-well weefsel cultuur polystyreen plaat...

Video: Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bioengineering

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Chemistry

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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