Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Nan Chen1,2, Kai Zhu1,2, Shiqiang Yan3,4, Junmin Li1,2, Tianyi Pan4, Mieradilijiang Abudupataer1,2, Fazle Alam4, Xiaoning Sun1,2, Li Wang3,4, Chunsheng Wang1,2
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Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

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Chen, N., Zhu, K., Yan, S., Li, J., Pan, T., Abudupataer, M., Alam, F., Sun, X., Wang, L., Wang, C. Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution. J. Vis. Exp. (159), e60920, doi:10.3791/60920 (2020).


During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.


Three-dimensional (3D) bioprinting has been a promising method to manufacture complex architectural and functional replicas of native tissues in biofabrication and regenerative medicine1,2,3. The common strategies of bioprinting, including inkjet, extrusion, and stereolithography printing, have pros and cons from different perspectives4. Among these techniques, the extrusion procedure is most commonly used due to its cost-effectiveness. Bioink plays a key role in the process stability of extrusion bioprinting. The ideal cell-laden bioink should not only be biocompatible but also be suitable for mechanical properties5. Bioinks with low viscosity are typically presented as a liquid-like state. These bioinks can be easily and quickly deposited and avoid cell membrane damage induced by high shear stress during extrusion. However, in complex cases requiring long-term printing periods, low viscosity often gives rise to the inevitable sedimentation of the encapsulated cells in the bioink reservoir, which is usually driven by gravity and leads to an inhomogeneous cell dispersion in the bioink6,7. Consequently, a bioink with inhomogeneous cell dispersity hampers the in vitro bioprinting of a functional tissue construct.

Several recent studies focusing on bioinks have reported the promotion of homogenous dispersity of encapsulated cells. A modified alginate bioink based on dual-stage crosslinking was used for extrusion bioprinting8. An alginate polymer was modified with peptides and proteins in this study. Cells presented a more homogeneous distribution in this modified alginate than in the commonly used alginate due to the attachment sites provided by the peptides and the proteins. Alternatively, blended bioinks have been utilized to solve the sedimentation of cells in bioink. A blended bioink containing polyethylene glycol (PEG) and gelatin or gelatin methacryloyl (GelMA) with improved mechanical robustness was used in another study9. The encapsulated cells presented a homogeneous distribution mainly because the viscosity of the blended bioink was improved. In general, there are several factors influencing the dispersity of the encapsulated cells in the bioink, such as the viscosity of the bioink, the gravity of the cells, the density of the cells, and the duration of the working period. Among these factors, the gravity of cells plays a critical role in promoting sedimentation. The buoyancy and friction provided by the viscous bioink have been investigated as the main forces against gravity to date10.

Herein, we developed a novel strategy to promote homogeneous dispersity of the encapsulated cells in bioink by manipulating multiple liquid interfaces in the bioink reservoir. These liquid interfaces created by the multilayered modification of bioink can not only provide interfacial retention, which retards the sedimentation of cells, but also maintain a suitable biocompatibility and rheological behavior of the bioink. In practice, we modified aqueous GelMA solution (5%, w/v) with silk fibroin (SF) in a multilayered manner to longitudinally produce four interfaces, providing interfacial tensions in the blended bioink. As a result, the gravity loading on the cells was offset by the man-made interfacial tension, and a nearly homogeneous dispersion of the encapsulated cells in the bioink was obtained due to less sedimentation across the adjacent layers of cells. No similar protocol to slow down the sedimentation of encapsulated cells by manipulating interfacial retention in liquid bioinks has been reported to date. We present our protocol here to demonstrate a new way to solve cell sedimentation in bioprinting.

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1. Preparation of cell-laden SF-GelMA

  1. Sterilize all the materials by using 0.22 μm syringe filter units. Perform all the steps in a biological safety cabinet.
  2. Warm 1x PBS to 50 °C, and dissolve gelatin in the heated 1x PBS with stirring. The final concentration of gelatin in PBS should be 10% (w/v).
  3. Add methacrylic anhydride into the gelatin solution (weight ratio of methacrylic anhydride to gelatin of 0.6 to 1) slowly with stirring, and mix the complex for at least 1 h (50 °C). Typically, prepare 200 mL of 10% gelatin solution with 12 g of methacrylic anhydride; the volume depends on the needs of the study.
  4. Transfer the mixed solution containing gelatin and methacrylic anhydride to a 50 mL sterile tube.
  5. Centrifuge the mixed solution at 3,500 x g. It usually takes 3-5 min to obtain two layers. Collect the upper layer (GelMA) and discard the bottom layer (unreacted methacrylic anhydride).
  6. Dilute the upper layer solution obtained in step 1.5 with two volumes of deionized water (40−50 °C).
  7. Dialyze the solution obtained in step 1.6 with a 12-14 kDa molecular weight cutoff dialysis membrane against deionized water for 5−7 days (40−50 °C). Change the water twice every day.
  8. Collect and freeze the GelMA solution at −80 °C overnight.
  9. Lyophilize the GelMA solution for 3−5 days in a freeze dryer with the temperature set to -45 °C and the pressure set to 0.2 mbar.
  10. Dissolve the lyophilized GelMA (degree of substitution of approximately 75%) in 1x PBS containing 10% FBS (fetal bovine serum, v/v), 25 mM HEPES (N-2-hydroxyethylpiperazine-N-ethane-sulfonic acid), and photoinitiator (0.5%, w/v) to obtain the GelMA bioink preparation.
    NOTE: The degree of substitution of GelMA can be calculated by a ninhydrin assay3.
  11. Mix the GelMA solution (10%, w/v) with different volumes of initial SF solution (5%, w/v) and different volumes of 1x PBS to obtain SF-GelMA bioinks with different concentrations of SF. The proportion per 1 mL of GelMA and SF solution in the different bioinks is presented in Table 1.
    NOTE: All bioinks must contain a final concentration of 5% (w/v) GelMA, but the concentration of SF varies: 0.5, 0.75, 1.0, 1.25, and 1.5% (w/v) in the different SF-GelMA bioinks. Use SF-M-Layered-GelMA to term the GelMA bioink modified with SF in a layer-by-layer manner. Use SF-X-GelMA to term those GelMA modified with SF in a homogeneous manner (e.g., GelMA with modification of 1% SF was termed SF-1-GelMA).
  12. Sonicate all bioinks for 10-20 min.
  13. Grow NIH3T3 cells using DMEM (Dulbecco's modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 °C with 5% CO2). Passage the cells at a ratio of 1:3 when the density reaches 80%.
  14. Centrifuge the suspension of NIH3T3 cells at 1500 rpm for 5 min. Remove the supernatant with suction and resuspend the cell pellet with fresh bioink solution in a 15 mL sterile tube.
    NOTE: The concentration of the encapsulated cells in the bioink should be 1 x 106 cells/mL, and the concentration needs to be calculated with a cytometer.
  15. Use 2 mL of different SF-GelMA bioinks to suspend the cell pellets to obtain various cell-laden bioinks.

2. Loading, reheating and bioprinting of the SF-M-Layered-GelMA

  1. Load 0.4 mL of cell-laden SF-0.5-GelMA into the bottom layer of the syringe.
    NOTE: Use a 2 mL syringe as the bioink reservoir in this study. Load different SF-GelMA bioinks into the syringe in a layer-by-layer manner.
  2. Place the syringe in ice water bath (0 °C) for 5 min to cause the bottom-layer bioink to transform into a gel state.
  3. Load 0.4 mL of cell-laden SF-0.75-GelMA bioink above the bottom layer.
  4. Place the syringe with the two layers of bioinks in an ice water bath (0 °C) for 5 min to cause the two layers of bioinks to transform into a gel state.
  5. Cycle the loading and cooling steps another 3 times with the remaining 3 kinds of bioinks (SF-1-GelMA, SF-1.25-GelMA, SF-1.5-GelMA) to obtain a multilayered bioink system with different concentrations of SF in the different layers. The volume used for the loading of all bioinks should be 0.4 mL.
  6. Reheat the multilayered bioink by placing the syringe in an incubator (37 °C) for 30 min prior to bioprinting.
  7. Use a 2 mL syringe as the bioink reservoir with a 27 G printing nozzle. Set the speed of flow at 50 μL/min, the moving speed of the nozzle at 2 mm/s, and the height of the nozzle at 1 mm. Perform the bioprinting procedure at room temperature (approximately 20 °C).
    1. Print the tissue construct in an extrusion manner using a custom-made bioprinter under the parameters adapted from our previous studies11,12.
  8. Use ultraviolet light (365 nm, 800 mW) for 40 s to crosslink the bioprinted tissue construct.
  9. Culture the crosslinked tissue construct in DMEM (Dulbecco's modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 °C with 5% CO2). The medium was changed every 8 h during the first 2 days and every 2-3 days thereafter.

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Representative Results

A schematic of the preparation of cell-laden bioinks is shown in Figure 1. After preparation of the different bioinks, loading, reheating and bioprinting were performed (Figure 2). To evaluate the distribution of the encapsulated cells in the bioink reservoir, a bioprinting procedure was performed using three different cell-laden bioinks in three 96-well plates (Figure 3A). Two control groups (pristine GelMA and SF-1-GelMA bioinks) and the experimental group (SF-M-Layered-GelMA bioink) were used to investigate the dispersity of the encapsulated cells (Figure 3B). Sixty microliters of cell-laden bioink was extruded in every well of the three 96-well plates. The total volume of the three kinds of bioink was 2 mL, and as a result, the period of bioprinting was more than 30 min. With the additional incubation prior to printing (30 min), the total working period was more than 1 h and was deemed a suitable fabrication duration for bioprinting large tissues and organs. The number of cells in the target wells, labeled in Figure 3A, in the three plates was counted. The counts of the cells in different wells could reflect the dispersity of the encapsulated cells among the different groups. The results showed that as the bioprinting procedure progressed, the density of cells in the target wells decreased in all groups. In the SF-0-GelMA group, approximately 70% of the cells were deposited in the bottom layer after the total procedure. In the SF-1-GelMA group, approximately 40% of the cells were deposited in the bottom layer, and approximately 5% of the cells were deposited in the top layer. In the SF-M-Layered-GelMA group, the deposition of encapsulated cells was more homogeneous than that of the control groups (Figure 3C and 3D).

Figure 1
Figure 1: Schematic of the SF-GelMA bioink preparation. The SF-GelMA was prepared by mixing the silk fibroin (SF) and GelMA/photoinitiator (PI) complex followed by ultrasonic treatment for 10-20 min. Then, the target cells in the SF-GelMA mixture were suspended to prepare the cell laden SF-GelMA bioink. Please click here to view a larger version of this figure.

Figure 2
Figure 2: The loading, reheating, and bioprinting procedure. The cell laden SF-GelMA, as the bioink (aq), was loaded into the bioink reservoir and transformed into the gel state from the cooling procedure. The loading and cooling procedure was repeated 5 times using the bioinks (aq) with different concentrations of SF (0.5, 0.75, 1.0, 1.25, and 1.5%) to obtain SF-Multilayered-GelMA (SF-M-Layered-GelMA). The gel state SF-M-Layered-GelMA was reheated by placing the bioink in an incubator for 30 min. The gel state bioink turned into a liquid state after incubation. Then, the prepared bioink was used for printing using a 2 mL syringe as the bioink reservoir with a 27 G printing nozzle. The printed tissue construct was crosslinked by using UV light. Aq means aqueous solution. Please click here to view a larger version of this figure.

Figure 3
Figure 3: The bioprinting procedure used three different cell-laden bioinks. (A), Sixty microliters of bioink was extruded into the corresponding 96-well plate per minute, and it took more than 30 min to achieve the bioprinting procedure in the 96-well plate. (B), Three different kinds of bioinks were used to investigate the dispersity of the encapsulated cells in the bioprinting procedure: two control groups (pristine GelMA and SF-1-GelMA bioinks) and the experiment group (SF-M-Layered-GelMA bioink). (C-D), Cell densities of the No. 1, 5, 10, 15, 20, 25, and 30 wells in the three plates were detected with staining, showing the distribution of encapsulated cells in the three bioinks, and the deposition of the encapsulated cells in the SF-M-Layered-GelMA group was more homogeneous. Please click here to view a larger version of this figure.

Bioinks Materials (ml)
SF-0.5-GelMA 0.5 0.1 0.4
SF-0.75-GelMA 0.5 0.15 0.35
SF-1.0-GelMA 0.5 0.2 0.3
SF-1.25-GelMA 0.5 0.25 0.25
SF-1.5-GelMA 0.5 0.3 0.2

Table 1: Preparation of SF-GelMA bioinks

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The stability of the multilayered system is a key point to perform this protocol successfully. We theoretically calculated the diffusion of SF molecules in the GelMA solution based on Nauman’s study13. It was found that the diffusion of proteins in solution was related to their molecular weight. The average molecular weight (MW) of bovine serum albumin (BSA) is 66.5 kDa, and its diffusion coefficient is 64-72 μm2/s. The average MW of fibrinogen is 339.7 kDa, and its diffusion coefficient is 23-34 μm2/s. The average MW of SF molecules in our study is approximately 100 kDa. Based on the results of Nauman’s study, the diffusion coefficient of a single SF molecule is between 23-72 μm2/s in water at 25 °C. According to the Stokes–Einstein equation, the diffusion coefficient is defined as follows:

Equation 1

where D is the diffusion coefficient, K is the consistency, T is the absolute temperature, η is the viscosity of the solution, and d is the diameter. It can be concluded that the difference in diffusion coefficient of SF molecules in water and in GelMA solution is determined by the difference in viscosity of these two surrounding solutions. Moreover, the diffusion radius can be calculated as follows:

Equation 2

where R is the diffusion radius, D is the diffusion coefficient, and T is the diffusion time. Pure water presents a viscosity at 8.9 x 10-4 Pa·s, and the viscosity of GelMA at zero shear rate in our study was higher than 1000 Pa·s. Hence, the diffusion radius of a single SF molecule at 25 °C is less than 0.66-1.18 μm/hour in the GelMA solution. This indicates that SF can hardly diffuse across the adjacent layers in the SF-M-Layered-GelMA system, which demonstrates the stability of the multilayered system.

The gravity-driven sedimentation of the encapsulated cells is inevitable when bioprinting with liquid-like bioinks. In our study, we developed a novel modification of low viscosity GelMA bioink with cyclic loading-cooling to retard the sedimentation of cells by creating interfacial retention. The multilayered interfacial retention is supposed to offset the action of gravity of the encapsulated cells and consequently prevent the sedimentation of the encapsulated cells across the adjacent layers in the bioink reservoir. The interfacial retention was presented longitudinally in the bioink reservoir between each adjacent layer due to the difference between the rheological behavior of different bioinks loaded in different layers. These interfacial tensions began to work as soon as the encapsulated cells sedimented to the interface. As a result, the gravity pull was offset, and sedimentation was stopped. Although this protocol is novel, more applications utilizing this protocol in other liquid-like bioink systems should be studied for promotion and optimization.

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The authors have nothing to disclose.


The authors acknowledge grants from the National Natural Science Foundation of China (81771971, 81970442, 81703470 and 81570422), National Key R&D Program of China (2018YFC1005002), Science and Technology Commission of Shanghai Municipality (17JC1400200), Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027).


Name Company Catalog Number Comments
2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (PI2959) TCI M64BK-QD
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Gibco 15630080
Dulbecco’s modified Eagle’s medium (DMEM) Gibco 10569044
fetal bovine serum (FBS) Gibco 10091
Gelatin Sigma-Aldrich V900863MSDS
Methacrylic anhydride (MA) Sigma-Aldrich 276685MSDS
Penicillin–streptomycin antibiotics Gibco 15140163
Phosphate-buffered saline (PBS) Gibco 10010049
Silk fibroin Advanced BioMatrix 5154



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