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 o...

<ol>
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B. 3 (7), 1268-1280 (2015).</li><li>Ding, F., 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=A+dynamic+and+self-crosslinked+polysaccharide+hydrogel+with+autonomous+self-healing+ability.">A dynamic and self-crosslinked polysaccharide hydrogel with autonomous self-healing ability.</a> Soft Matter. 11 (20), 3971-3976 (2015).</li><li>Wei, Z., 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=Self-healing+gels+based+on+constitutional+dynamic+chemistry+and+their+potential+applications.">Self-healing gels based on constitutional dynamic chemistry and their potential applications.</a> Chem. Soc. Rev. 43 (23), 8114-8131 (2014).</li><li>Li, Y., 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=Modulus-regulated+3D-cell+proliferation+in+an+injectable+self-healing+hydrogel.">Modulus-regulated 3D-cell proliferation in an injectable self-healing hydrogel.</a> Colloid. Surface. B. 149, 168-173 (2017).</li><li>Tseng, T. C., 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=An+Injectable,+Self&#8208;Healing+Hydrogel+to+Repair+the+Central+Nervous+System.">An Injectable, Self&#8208;Healing Hydrogel to Repair the Central Nervous System.</a> Adv. Mater. 27 (23), 3518-3524 (2015).</li><li>Yu, L., Ding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+hydrogels+as+unique+biomedical+materials.">Injectable hydrogels as unique biomedical materials.</a> Chem. Soc. Rev. 37 (8), 1473-1481 (2008).</li><li>Yang, L., 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=Improving+Tumor+Chemotherapy+Effect+by+Using+an+Injectable+Self-healing+Hydrogel+as+Drug+Carrier.">Improving Tumor Chemotherapy Effect by Using an Injectable Self-healing Hydrogel as Drug Carrier.</a> Polym. Chem. , (2017).</li><li>Zhang, Y., 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=A+magnetic+self-healing+hydrogel.">A magnetic self-healing hydrogel.</a> Chem. Commun. 48 (74), 9305-9307 (2012).</li><li>Zhang, Y., 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=Synthesis+of+an+injectable,+self-healable+and+dual+responsive+hydrogel+for+drug+delivery+and+3D+cell+cultivation.">Synthesis of an injectable, self-healable and dual responsive hydrogel for drug delivery and 3D cell cultivation.</a> Polym. Chem. 8 (3), 537-534 (2017).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat. Mater. 13 (6), 645-652 (2014).</li><li>Geerligs, M., Peters, G. W., Ackermans, P. A., Oomens, C. 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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 border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 c...

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 ...

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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18.8K vistas.  Tsinghua University. 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 hy...