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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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 prepared using different solid contents and ratios of glycol chitosan and DF PEG. Homogeneous hydrogels were quickly formed in minutes by mixing gelation solutions under room temperature, though the gelation speed could also slow down by dilute solutions. Rheology test was employed to evaluate the mechanical strengths of different hydrogels. The storage moduli (G&#39;) of those hydrogels were found to approximately differ from 900 Pa (soft), 2,100 Pa (medium) to 4,700 Pa (stiff) (Table 1 and Figure 2A). This is contributed to the higher crosslinking density by adding more polymer gelators in the hydrogel network, resulting in higher storage modulus (greater stiffness). The self-healable property is also confirmed by recovery of the hydrogel&#39;s modulus (Figure 2B). It is known that ECM stiffness differs for various tissues, thus the hydrogel could offer adjustable mechanical cues and meet different research requirements by varied ratios between glycol chitosan and DF PEG polymer gelators18.
L929 cells, a typical mouse fibroblast cell line with in vivo environment tissue stiffness of ~ 5,600 pa19, was used as a cell model to be embedded in the hydrogel. After the cell encapsulation in hydrogels, it can be easily observed that the cells in the hydrogels showed extremely high viability (&#62;99%) throughout the culture process, confirming the excellent biocompatibility of the chitosan-based hydrogel. A remarkable increase of cell density in the hydrogel implied that this hydrogel could support proliferation of the L929 cells even without extra-added growth factors (Figure 3A). After 7 days in culture in the hydrogel, the cell number increased 300% (Figure 3B). It is reported that cells cultured in scaffolds could differentiate following mechanical cues20,21, which might imply further research using this 3D hydrogel culture method.
Implanting cells with injectable hydrogels has gained incomparable advantages to greatly enhance the delivery efficiency and viability of the implanted cells14. Within a dynamic imine crosslinked network, the hydrogel could self-heal after injection. The self-healable property enables the hydrogel to be injectable, which implies potential applications for the hydrogels as injectable multiple carriers. To further evaluate this hydrogel as an injectable cell carrier, an injection-mimicking and post-culturing experiment was performed. The cell embedded hydrogel was loaded in a syringe and squeezed out through a 48 G needle into a Petri dish, followed by a 7 day post-culturing process with cell viability and proliferation rate studied.
As shown in Figure 4A, the squished hydrogel pieces could self-heal to reform an integrated hydrogel about 1 h after injection, which is helpful in cell delivery. For cells in the hydrogel, when compared with the image took just after encapsulation (Figure 4A, left insert), the cell density increased dramatically after 7 days (Figure 4A, middle insert). In the enlarged vision, a more detailed cell-fissional process in which some cells are found to be divided into two (Figure 4A, right insert), directly confirmed by 3D cell proliferation in the hydrogel. Figure 4B shows the confocal images of the live-dead cell assays after injection and post-culture experiment. The density of the injected cells increased obviously with a high cell viability (&#62;99%), demonstrating the successful cell proliferation in the 3D hydrogel after injection. To learn whether the injection affected the cell proliferation rate, a comparison of quantitative statistics analysis for the cell proliferation rates in hydrogels with or without injection was performed (Figure 4C). The cell proliferation rate after injection, although slightly decreased, remained in a high level (~75%), and the cell number increased 145% relatively after 7 days in culture in the hydrogel, indicating that the shearing force during injection has an observable limited negative influence on the cell proliferation.
We described a typical procedure of this hydrogel application but researchers can modify the protocol to fit specific research requirements. Polymer gelators have significant influences on the hydrogel and are easily modifiable. For example, the stiffness of this hydrogel could be easily manipulated by different ratios of glycol chitosan and DF PEG gelator. Meanwhile, the use DF PEG of other molecular weights could also fulfill this target. We used PEG 4K in this protocol, but PEG 2K to 10K may also work. When using other PEGs, it is important to monitor the gelation time and stiffness specifically. Other functional polymer gelators may also work if the researchers have synthesis skills to prepare specific functional benzaldehyde-terminated polymers17.
This protocol has several limitations. Firstly, due to the dynamic structure, the stiffness of this hydrogel is limited compared with a covalent bond crosslinked hydrogel. One may encounter failures to prepare hydrogels with too high of a modulus design. Secondly, this hydrogel is bio-degradable, thus limiting the long-term cell culture within the hydrogel. Thirdly, the hydrogel offers a 3D crosslinked cultivation environment that has diffusion concerns. To date, most of the bio-analyses are based on 2D culture. The 3D structure might hinder further cell related in vivo studies.
When using this protocol, there are several critical steps to follow. Firstly, during synthesis of the polymer gelator DF PEG, dehydration is very important. Secondly, when performing cell-related procedures, sterile operation is critical. Thirdly, the staining time should be well controlled in the hydrogels to get nice confocal images.
In summary, the protocol introduced a facile preparation of chitosan-based injectable hydrogels, which are applied as a platform for 3D cell culture and could offer adjustable mechanical cues for various researches. It has already been applied in areas regarding drug delivery, cell therapy, and tumor chemotherapy, which not only is a testament to its performance, but also increases its potential for biomedical applications.

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

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&#39;s contents and ratios prepared with different mechanical strengths is summarized in Table 1. The hydrogel&#39;s self-healable and rheology property presents the hydrogel&#39;s stiffness by storage modulus versus frequency test in Figure 2. The cell confocal images and cell numbers with days of culture in hydrogels are presented in Figure 3, confirming the cell viability and cell proliferation via 3D cell culture. Figure 4 presents microscopy, confocal images, and analysis of cell proliferation rates for cells embedded in hydrogels that suffered injection and post-culture. High cell viability and cell proliferation indicate that the cells embedded in the hydrogel suffered no destructive harm and could recover by post-culture, indicating the hydrogel&#39;s injectability.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56253/56253fig1.jpg" /> 
Figure 1: Preparation of the hydrogel and its use as 3D cell culture. (A) Hydrogel preparation; (B) Cell suspension preparation; (C) 3D cell culture in hydrogels with or without injection. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig1large.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/56253/56253fig2.jpg" /> 
Figure 2: Hydrogel stiffness. Storage modulus G&#39; versus frequency of (A) original and (B) self-healed hydrogels. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig2large.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/56253/56253fig3.jpg" /> 
Figure 3: 3D cell culture in the hydrogel. (A) 3D confocal images of L929 cells embedded in stiff strength hydrogel (Green: live cells; Red: dead cells); (B) Cell counting results of L929 cells embedded in the hydrogel after cell encapsulation at denoted time. Scale bar = 300 &#181;m. Data presented as Mean &#177; SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig3large.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/56253/56253fig4.jpg" /> 
Figure 4: 3D cell post-culture in hydrogel after injection. (A) A schematic image of injection and post-culture of a cell-loaded hydrogel. Insert: Microscopy images of cells embedded in hydrogels before injection (left) and after cell post-culture for 7 days (middle and right). Scale bar = 100 &#181;m; (B) Confocal images of L929 cells embedded in a stiff strength hydrogel and post-cultured after injection (Green: live cells; Red: dead cells), Scale bar = 300 &#181;m; (C) A comparison of the proliferation rate of cells cultured in hydrogels with or without injection after encapsulation. Data presented as Mean &#177; SD. (Adapted from reference12; Copyright &#169; 2017 Elsevier). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56253/56253fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydrogel Stiffness&#160;</td>
			<td>Soft</td>
			<td>Medium</td>
			<td>Stiff</td>
		</tr>
		<tr>
			<td>Glycol chitosan (mg)</td>
			<td>6.6</td>
			<td>6.6</td>
			<td>6.6</td>
		</tr>
		<tr>
			<td>DF PEG (mg)</td>
			<td>4.4</td>
			<td>8.8</td>
			<td>17.6</td>
		</tr>
		<tr>
			<td>Polymer gelator content (wt%)</td>
			<td>1</td>
			<td>2</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Deionized water (mL)</td>
			<td>0.4</td>
			<td>0.4</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>Gelaion time (min)</td>
			<td>7.5</td>
			<td>5</td>
			<td>3.5</td>
		</tr>
		<tr>
			<td>G&#8217; (Pa)*</td>
			<td>900</td>
			<td>2100</td>
			<td>4700</td>
		</tr>
		<tr>
			<td>* tested under frequency 6.3 rad s-1, strain 1% using a parallel plate (diameter 20 mm) on a rotational rheometer.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Information of hydrogels prepared with different mechanical strengths.

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

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

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 ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Nanyang Technological University

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Chemistry

19.0K Views.  Tsinghua University. Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel’s mechanical strength and its application in 3D cell culture are presented.

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

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

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

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.

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochemistry

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

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Biology

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.

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) ...

Developmental Biology

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

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.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

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.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

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.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

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, ...

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

Summary

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Chapters in this video

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

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

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

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