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

Summary

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.

Abstract

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.

Introduction

Hydrogels are crosslinked polymer materials with large amounts of water and soft mechanical properties, and they have been used in many bio-medical applications^1,2. 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 culture^3,4. 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 ass....

Protocol

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

1. Synthesis of benzaldehyde terminated di-functionalized polyethylene glycol (DF PEG)
   1. Pre-desiccation of PEG polymer
      1. Weigh 4.00 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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