Name: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture (Video) | JoVE Journal
Uploaded: 2017-09-29T14:00:00.000+00:00
Duration: 8 min 5 s
Description: Learn to prepare injectable chitosan-based hydrogels using dynamic imine chemistry for 3D cell culture applications. This method enables adjustable mechanical properties and self-healing capabilities for cell therapy research.

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5 minutes

This research was supported by the National Science Foundation of China (21474057 and 21604076).

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate safety practices when performing chemistry experiments, including the use of a fume hood and personal protective equipment (safety glasses, protective gloves, lab coat, etc.). The protocol requires standard cell handling techniques (sterilizing, cell recovery, cell passaging, cell freezing, cell staining, etc.).
1. Preparation of Hydrogels
<ol>
	<li>Synthesis of benzaldehyde terminated di-functionalized polyethylene glycol (DF PEG)
	<ol>
		<li>Pre-desiccation of PEG polymer
		<ol>
			<li>Weigh 4.00 g PEG (MW 4,000, 1.00 mmol), transfer it into a round bottom flask (250 mL), and add toluene (100 mL). 
			Note: Other PEGs with different molecular weights can work for the hydrogel formation but will lead to different stiffness.</li>
			<li>Heat the solution by a heat gun (or a hot plate around 40 &#176;C) mildly to help dissolve all the polymers. After all the polymers dissolve, use an evaporator to remove all the solvents. Repeat this dissolve and dry process two times to make the dried PEG polymers. 
			CAUTION: This should be performed in a fume hood with extreme care. Toluene is flammable.</li>
		</ol>
		</li>
		<li>Benzaldehyde termination reaction of PEG
		<ol>
			<li>Add a magnetic stirrer and tetrahydrofuran (THF, 100 mL) to the flask and dissolve PEG using a stirrer. Add 4-carboxybenzaldehyde (0.90 g, 6.0 mmol) and 4-dimethylaminopyridine (0.07 g, 0.6 mmol) sequentially to the solutions to dissolve all the solids completely.</li>
			<li>Add N,N&#39;-dicyclohexylcarbodiimide (DCC, 1.25 g, 6.0 mmol) to the solution, and add a drying tube that is filled with anhydrous CaCl2 to the flask. Keep the reaction under stirring at room temperature (~ 20 &#176;C) for around 12 h.</li>
		</ol>
		</li>
		<li>Post-reaction process
		<ol>
			<li>Filter out the white solids generated in the solution by vacuum after the reaction is finished. Pour the solution into cold diethyl ether (500 mL) under stirring to precipitate the white solids. Gather all the white solids by filter and dry the solids in a fume hood.</li>
			<li>Dissolve the white solids into THF again, filter out any insoluble white solids, pour the solution into fresh cold diethyl ether to precipitate the white solids, and dry it. Repeat this process two to three times, and then place the white solids in a vacuum drying oven (20 &#176;C, 0.1 mbar) to dry them completely. Collect the white powder as the final product: benzaldehyde terminated DF PEG.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of hydrogels
	<ol>
		<li>Weigh different amounts of DF PEG (0.11 g, 0.028 mmol; 0.22 g, 0.055 mmol; 0.44 g, 0.110 mmol) in a tube (10 mL), add deionized water (5.0 mL), and use a vortex or magnetic stirrer to help dissolve the polymer.</li>
		<li>Dissolve glycol chitosan (0.495 g, 6.18 x 10-3 mmol) in deionized water (15.0 mL) in a tube (50 mL), and use a vortex for a few minutes to homogenize the chitosan solution (3 wt%).</li>
		<li>Add the glycol chitosan solution (0.2 mL, 3 wt%) and DF PEG solutions (0.2 mL) sequentially in a tube (2.0 mL). Use a vortex to mix the solutions homogeneously to form hydrogels in several minutes. Follow the ratios in Table 1 to make hydrogels of different mechanical strengths. 
		Note: Use the tube-inverting method to determine whether the hydrogel has already formed.</li>
	</ol>
	</li>
	<li>Rheology analyses
	<ol>
		<li>Carry out rheology analyses on a rotational rheometer with a parallel steel plate (diameter: 20 mm). For the gelation test, spread glycol chitosan solution (0.2 mL, 3 wt%) on a lower plate. Then, add DF PEG aqueous solutions (0.2 mL, 2 wt%) evenly dropwise onto the chitosan solution surface and mix with a pipette quickly. Lower down the upper plate and begin to test.</li>
		<li>For hydrogel&#39;s stiffness test, cut a hydrogel into a circle (diameter: 20 mm) and measure the storage modulus (G&#39;) values versus frequency analyses at 1% strain. Typical G&#39; values at 6.3 rad s&#8722;1 are listed in Table 1. 
		Note: Carry out the rheology analyses of the hydrogel 0.5 h after the hydrogel formation to ensure dynamic bond stabilization of the hydrogel.</li>
		<li>For the hydrogel&#39;s self-healable property test, cut a hydrogel to pieces and gather the pieces together to self-heal to an integral piece. Then cut the hydrogel piece to make a circle (diameter: 20 mm) and put it on the rheometer to carry out the rheology analysis.</li>
	</ol>
	</li>
</ol>
2. 3D Cell Cultivation in Hydrogels
<ol>
	<li>Preparation of hydrogel gelation solutions
	<ol>
		<li>Weigh DF PEG (0.44 g, 0.11 mmol) in a sterile tube (4.0 mL), add in cell culture media (RPMI-1640, 2.0 mL), and use a vortex or stirrer to help dissolve the polymer to obtain the polymer solution (20 wt%). Load the solution in a syringe (10.0 mL) and then sterilize it by passing it through a micron bacteria-retentive filter (0.22 &#181;m).</li>
		<li>Weigh the glycol chitosan (0.165 g, 2.06 x 10-3 mmol) in a sterile tube (15.0 mL), add in cell culture media (RPMI-1640, 4.0 mL), and vortex to help dissolve the polymer to obtain the glycol chitosan solution (4.0 wt%). Load the solution in a syringe (10.0 mL) and filter it with a 0.22 &#181;m bacteria-retentive filter.</li>
	</ol>
	</li>
	<li>Cell cultivation in hydrogels 
	CAUTION: Perform all cell related procedures in a tissue culture hood. Basic knowledge of sterile technique is expected.
	<ol>
		<li>Preparation of cell suspension
		<ol>
			<li>Culture L929 cells in RPMI-1640 medium supplemented with 10% FBS, 5% penicillin (10 mL) in a Petri dish (diameter 10 cm), and incubate at 37 &#176;C, 5% CO2. Change the medium every day before use.</li>
			<li>Harvest the L929 cells with PBS containing trypsin (0.025 w/v %) and EDTA (0.01% w/v), then centrifuge (70 x g, 5 min) and re-suspend the cells in the RPMI-1640 medium (1.0 mL). Perform the cell counting using standard operation of blood-counting board. Re-suspend the cells to adjust the cell concentration to ~ 3.75 &#215; 106 cells/mL.</li>
		</ol>
		</li>
		<li>Cell encapsulation in hydrogels
		<ol>
			<li>Mix the L929 cell suspensions (0.4 mL, 3.75 x 106 cells mL-1) with glycol chitosan solution (0.4 mL) in a tube (4.0 mL) by vortex. Pipette the L929/glycol chitosan solution (0.8 mL) into the center of a confocal Petri dish (diameter 2.0 cm). Pipette the DF PEG solutions (0.2 mL) into the same dish, and gently pipette to mix the solution and induce hydrogel formation. 
			Note: Assess the hydrogel formation by tilting the petri dish.</li>
			<li>For direct cell culture, add additional amounts of RPMI-1640 culture media (1.0 mL) on top of the hydrogel. Put the cell embedded hydrogels (1.0 mL, 1.5 wt% glycol chitosan, 4.0 wt% DF PEG, 1.5 &#215; 106 cells mL-1) in an incubator (37 &#176;C, 5% CO2) and change the medium every day. Prepare for cell imaging on Day 1, 3, 5, and 7 after cell encapsulation.</li>
		</ol>
		</li>
		<li>For the 3D cell post-culture in hydrogels after injection, prepare cell loaded hydrogel (1.0 mL, see step 2.2.2) in a syringe (10.0 mL, 48 G needle). After the hydrogel forms, inject the hydrogel slowly into a Petri dish for confocal imaging. Add an additional amount of culture media (1.0 mL) on top of the hydrogel and change it every day. Put the Petri dish in an incubator (37 &#176;C, 5% CO2) and prepare for imaging afterwards. 
		CAUTION: Please check and follow the safety operation protocol of a syringe.</li>
	</ol>
	</li>
	<li>Cell viability analysis
	<ol>
		<li>Confocal observation
		<ol>
			<li>Rinse the hydrogels with PBS (1.0 mL) for two times. Stain the hydrogels with Fluorescein diacetate (FDA, 0.5 mL, 0.05 mg/mL) and propidium iodide (PI, 0.5 mL, 0.08 mg/mL) solutions for 15 min. After staining, remove all the solvents.</li>
			<li>Observe the hydrogels using a confocal microscope under excitation wavelengths of 488 nm and 543 nm to visualize live and dead cells, respectively. Take z-stacks through every 2 &#181;m depth of the hydrogels to validate an even distribution of cells throughout. 
			Note: FDA stains live cells while PI stains dead cells.</li>
		</ol>
		</li>
		<li>Degrade the hydrogel (1.0 mL) with acetic acid (HAc, 3 v%, 1.0 mL) for 5 min and pipette into a tube (4.0 mL). Collect cells by centrifuge (70 x g, 5 min) and re-suspend the cells in RPMI-1640 cell culture medium (1.0 mL). Perform cell counting using a blood counting board.</li>
	</ol>
	</li>
</ol>

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<li>Tseng, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Injectable%2C+Self%E2%80%90Healing+Hydrogel+to+Repair+the+Central+Nervous+System%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">An Injectable, Self‐Healing Hydrogel to Repair the Central Nervous System.</a> Adv. Mater. 27 (23), 3518-3524 (2015).</li>
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<li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+memory+and+dosing+influence+stem+cell+fate%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">Mechanical memory and dosing influence stem cell fate.</a> Nat. Mater. 13 (6), 645-652 (2014).</li>
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The authors have nothing to disclose.

The hydrogel presented in this protocol (Figure 1) has two main components: the natural polymer glycol chitosan and a synthetic benzaldehyde terminated polymer gelator DF PEG, which are both biocompatible materials. Synthesis of DF PEG is presented using a one-step modification reaction. PEG of molecular weight 4,000 was chosen in this protocol in concerns of solubility, modification efficiency, as well as hydrogel stiffness. A series of hydrogels with different mechanical strengths were pre...

Hydrogels are crosslinked polymer materials with large amounts of water and soft mechanical properties, and they have been used in many bio-medical applications1,2. Hydrogels can offer a soft and wet environment, which is very similar to the physiological surroundings for cells in vivo. Therefore, hydrogels have become one of the most popular scaffolds for 3D cell culture3,4. Compared to 2D Petri dish cell culture, 3D cell culture has advanced quickly to offer an extracellular matrix (ECM) mimicked microenvironment for cells to contact and assemble for proliferation and differentiation purposes5. Additionally, hydrogels containing natural polymers could offer bio-compatible and promoting environments for cells to proliferate and differentiate3. Hydrogels derived from synthetic polymers are preferred for their simple and clear components, which exclude complex influences like animal-origin proteins or viruses. Among all the hydrogel candidates for 3D cell culture, hydrogels that are easily prepared and have a consistent property are always preferred. The facility to adjust the hydrogel&#39;s properties to fit different research requirements is important as well6.
Here we introduce a facile preparation of a glycol chitosan-based hydrogel using dynamic imine chemistry, which becomes a versatile hydrogel platform for 3D cell culture7. In this method, well-known bio-compatible glycol chitosan are used to establish frames of the hydrogel&#39;s networks. Its amino groups are reacted with a benzaldehyde terminated polyethylene glycol as the polymer gelator to form dynamic imine bonds as crosslinkages of hydrogels8. Dynamic imine bonds can form and decompose reversibly and responsively to surroundings, endowing the hydrogels with mechanically adjustable crosslinked networks9,10,11. Due to its high water contents, bio-compatible materials, and adjustable mechanical strengths, the hydrogel is successfully applied as a scaffold for L929 cells in 3D cell culture12,13. The protocol here details the procedures, including polymer gelator synthesis, hydrogel preparation, cell embedding, and 3D cell culturing.
The hydrogel also shows several other features due to its dynamic imine crosslinkages, including its multi-responsive to various bio-stimuli (acid/pH, vitamin B6 derivative pyridoxal, protein papain, etc.), indicating that the hydrogel could be induced to decompose under physiological conditions8. The hydrogel is also self-healable and injectable, which means the hydrogel could be administrated via a minimal invasive injection method and gain an advantage in drug and cell deliveries14,15. By adding functional additives or specific predesigned polymer gelators, the hydrogel is compatible to gaining specific properties like magnetic, temperature, pH responsive, etc.16,17, which could fulfill a wide range of research requirements. Those properties reveal the hydrogel&#39;s potential capacity to be an injectable multiple carriers for drugs and cells in both in vitro and in vivo bio-medical research and applications.

<table><tbody><tr><td>Glycol chitosan</td><td>Wako Pure Chemical Industries</td><td>39280-86-9</td><td>90% degree of deacetylation</td></tr><tr><td>4-Carboxybenzaldehyde</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>619-66-9</td><td>99%</td></tr><tr><td>N, N&#039;-dicyclohexylcarbodiimide</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>538-75-0</td><td>99%</td></tr><tr><td>Calcium chloride anhydrous</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>10043-52-4</td><td>96%</td></tr><tr><td>4-dimethylamiopryidine</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>1122-58</td><td>99%</td></tr><tr><td>Polyethyleneglycol</td><td>Sino-pharm Chemical Reagent</td><td>5254-43-7</td><td>99%</td></tr><tr><td>Tetrahydrofuran</td><td>Sino-pharm Chemical Reagent</td><td>109-99-9</td><td>99%</td></tr><tr><td>Toluene</td><td>Sino-pharm Chemical Reagent</td><td>108-88-3</td><td>99%</td></tr><tr><td>Ethyl ether</td><td>Sino-pharm Chemical Reagent</td><td>60-29-7</td><td>99%</td></tr><tr><td>Acetic acid</td><td>Sino-pharm Chemical Reagent</td><td>64-19-7</td><td>99%</td></tr><tr><td>Anhydrous CaCl2</td><td>Sino-pharm Chemical Reagent</td><td>10043-52-4</td><td>99%</td></tr><tr><td>Fluorescein diacetate</td><td>Sigma</td><td>596-09-8</td><td>99%</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>25535-16-4</td><td>94%</td></tr><tr><td>RPMI-1640 culture media</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td></td><td>0.25%</td></tr><tr><td>PBS</td><td>Solarbio</td><td></td><td>0.01 M</td></tr><tr><td>Penicillin streptomycin solution</td><td>Hyclone</td><td></td><td>10,000 U/mL</td></tr><tr><td>Rheometer</td><td>TA Instrument</td><td></td><td>AR-G2</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>710-3channel</td></tr><tr><td>L929 Cells</td><td>ATCC</td><td></td><td>NCTC clone 929; L cell, L929, derivative of Strain L</td></tr><tr><td>Evaporator</td><td>EYELA</td><td></td><td>N-1100</td></tr><tr><td>48 guage needle</td><td>ShanghaiZhiyu Medical Material Co., LTD</td><td></td><td>48-guage</td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>DM3000 B</td></tr><tr><td>Microscope software</td><td>Imaris</td><td></td><td></td></tr><tr><td>Heat gun</td><td>Confu</td><td></td><td>KF-5843&amp;nbsp;</td></tr><tr><td>Petri dish</td><td>NEST</td><td></td><td></td></tr></tbody></table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

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.

A schematic presentation of this protocol on hydrogel preparation and its use as 3D cell culture is offered in Figure 1. Information of the hydrogel's contents and ratios prepared with different mechanical strengths is summarized in Table 1. The hydrogel's self-healable and rheology property presents the hydrogel's stiffness by storage modulus versus frequency test in Figure 2. The cell confocal images and cell numbers with days of culture in hy...

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 ...

The overall goal of this protocol is to prepare an injectable chitosan hydrogel for three-dimensional cell culture to ultimately use in therapeutic treatments. This method can answer question in the field of self-healing hydrogel, such as how to make a self-healing hydrogel by immersing whole way and how to use it for 3D cell culture. The my advantage of this technique is, by compatible self-healing hydrogel can be prepared by a very simple way, and the use of a 3D cell culture.This hydrogel is also injectable, so it can be used as a carrier for cell therapy. Now Mr.Yongsan Li will show you how to make the hydrogel and how to do the 3D cell culture using this hydrogel. Yongsan Li is our graduate student of our lab.To make the hydrogels, first synthesize DF PEG. In a fume hood weigh out four grams of molecular-weight 4, 000 PEG into a round-bottom flask, and add 100 milliliters of toluene. Then, to dissolve the polymers, warm up the solution.After all the polymers dissolve, use an evaporator to remove all the solvents. Then, repeat the process of mixing in toluene and evaporating it twice more to prepare the dry PEG polymers. Next, using a magnetic stirrer and 100 milliliters of DHF, dissolve the polymers.Then, dissolve 0.9 grams of 4-Carboxybenzaldehyde into the solution. Next, dissolve 0.07 grams of dimethylaminopyrimide into the solution. Now add 1.25 grams of DCC in a drying tube filled with anhydrous calcium chloride.Now allow the reaction to stir at room temperature for around 12 hours. The next day, use 500 milliliters of cold diethyl ether to precipitate the white solids, and use vacuum filtration to collect the solids. Discard the solute, and dry the solids under good ventilation.Next, dissolve the white solids in 100 milliliters of THF. Any insoluble white solids must be filtered out before proceeding. Now precipitate the dissolved solids using fresh cold diethyl ether.Then use vacuum filtration to collect the solids, and let them dry out again. Repeat this process two to three times, and then place the white solids in a vacuum-drying oven to dry them completely. The resulting white powder is the final product:benzaldehyde-terminated DF PEG.To encapsulate the cells in the hydrogel for culturing, first dissolve 0.44 grams of DF PEG into two milliliters of cell-growth media in a four-milliliter tube. Vortex the mixture into a 20%solution by weight. Next, sterilize the solution.Load it into a 10-milliliter syringe, and press it through a 0.22-micron bacteria-retentive filter. Now mix 0.165 grams of the glycol-chitosan with four milliliters of cell-culture media, using a vortex to make a 4%by-weight chitosan solution. Then, sterilize the solution using another 0.22-micron bacteria-retentive filter.Next, harvest the cells, such as L929 cells using trypsin. Wash the collected cells, and resuspend them in media at 3.75 million cells per milliliter. In the following step the cells are planted into a 3D matrix.Following these steps precisely is critical to produce success. Now mix 0.4 milliliters of the cell suspension with 0.4 milliliters of chitosan solution using a vortex. Pipet this mixture into the center of a two-centimeter-diameter petri dish, and pipet 0.2 milliliters of the DF PEG solution into the dish.Then, use gentle pipeting to induce the formation of the hydrogel. Assess the hydrogel's formation by tilting the dish and observing the solution's viscosity. To test the injectability of the encapsulated cells, make the same preparation in a 10-milliliter syringe.Then, after the hydrogel forms, slowly expel the hydrogel through a 48-gauge needle into the petri dish. Next, add one milliliter of culture media to the hydrogel-cell mixture in the plate, and transfer the plate to an incubator for culturing. Every other day, change the media, overlaying the 3D hydrogel cell culture.To prepare the cells encapsulated in hydrogels for imaging, first rinse the hydrogels with one milliliter of PBS two times. Then, stain the hydrogels with fluorescein-diacetate solution and propidium iodide solution. Overlay 0.5 milliliters of both solutions onto the hydrogel, and incubate the hydrogel for 15 minutes at room temperature.After the brief incubation with the stain solutions, aspirate away all the solvents. Now observe the hydrogels using confocal microscopy, with excitation at 488 nanometers to view living cells, and excitation at 543 nanometers to view dead cells. Collect image stacks with two-micron slices to validate the distribution of cells in the hydrogel.L929 cells. A typical mouse-fiberglass cell line with an in-vivo environment tissue stiffness of about 5, 600 Pascals were encapsulated in a hydrogel. Their viability was extremely high throughout the culture process, and they achieved a remarkable cell density.After seven days of culture in the hydrogel, the cell number increased by 200%Hydrogels that were expelled onto their cell-culture dish through a 48-gauge needle could self-heal to reform an integrated hydrogel after about one hour. After seven days in culture, these cells also proliferated dramatically. Evidence could be found of cell-fission events in which some cells appeared divided into two.The live-dead assay showed very high viability, demonstrating the successful cell proliferation in the 3D hydrogel after injection. This cell-proliferation rate was about 3/4 that of uninjected cells after seven days of culturing. So, although the sheering force during injection has a measurable negative effect, the injected cells were still quite proliferative.After watching this video you should know how to prepare a self-healing hydrogel and how to use this hydrogel for 3D cell culture and how delivery the cell with this hydrogel. Once mastered, this technique can be done within one hour if performed properly. Following best procedure, other method, like in-vivo injection for cell therapy or drug delivery, can be performed.

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Research

JoVE Journal

Chemistry

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

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Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

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3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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.

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

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

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.

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

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.

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

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, and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

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

Learn to prepare injectable chitosan-based hydrogels using dynamic imine chemistry for 3D cell culture applications. This method enables adjustable mechanical properties and self-healing capabilities for cell therapy research.

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

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