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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 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'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'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 networks^9,10,11. 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 culture^12,13. 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 deliveries^14,15. 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'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.

Protokół

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 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.
      2. Heat the solution by a heat gun (or a hot plate around 40 °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.
   2. Benzaldehyde termination reaction of PEG
      1. 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.
      2. Add N,N'-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 °C) for around 12 h.
   3. Post-reaction process
      1. 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.
      2. 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 °C, 0.1 mbar) to dry them completely. Collect the white powder as the final product: benzaldehyde terminated DF PEG.
2. Preparation of hydrogels
   1. 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.
   2. 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%).
   3. 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.
3. Rheology analyses
   1. 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.
   2. For hydrogel's stiffness test, cut a hydrogel into a circle (diameter: 20 mm) and measure the storage modulus (G') values versus frequency analyses at 1% strain. Typical G' values at 6.3 rad s⁻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.
   3. For the hydrogel'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.

2. 3D Cell Cultivation in Hydrogels

1. Preparation of hydrogel gelation solutions
   1. 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 µm).
   2. 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 µm bacteria-retentive filter.
2. Cell cultivation in hydrogels
   CAUTION: Perform all cell related procedures in a tissue culture hood. Basic knowledge of sterile technique is expected.
   1. Preparation of cell suspension
      1. 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 °C, 5% CO₂. Change the medium every day before use.
      2. 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 × 10⁶ cells/mL.
   2. Cell encapsulation in hydrogels
      1. 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.
      2. 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 × 10⁶ cells mL^-1) in an incubator (37 °C, 5% CO₂) and change the medium every day. Prepare for cell imaging on Day 1, 3, 5, and 7 after cell encapsulation.
   3. 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 °C, 5% CO₂) and prepare for imaging afterwards.
      CAUTION: Please check and follow the safety operation protocol of a syringe.
3. Cell viability analysis
   1. Confocal observation
      1. 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.
      2. 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 µm depth of the hydrogels to validate an even distribution of cells throughout.
         Note: FDA stains live cells while PI stains dead cells.
   2. 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.

Wyniki

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 c...

Dyskusje

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...

Materiały

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