- Email: [email protected]
A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation
Accepted Manuscript A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang PII: DOI: Reference:
S0167-577X(19)30611-1 https://doi.org/10.1016/j.matlet.2019.04.059 MLBLUE 26042
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
23 January 2019 11 April 2019 14 April 2019
Please cite this article as: J. Liu, Q. Wang, Y. Sun, H. Lin, J. Liang, Y. Fan, X. Zhang, A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation, Materials Letters (2019), doi: https://doi.org/10.1016/j.matlet.2019.04.059
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China Corresponding author: * Qiguang Wang
email: [email protected]
Keywords Composite materials, Multilayer structure, Particles Abstract Coculture models have long been used in cell and tissue engineering studies. The direct contact model normally offers excellent cell-cell interactions, but difficult to separate the two cell types after coculture. The non-contact model offers a good cell separation, but the long cell-cell distance limited the cell interactions. This study proposed a conceptual 3D coculture model based on collagen hydrogel microspheres (CHMs) with removable superparamagnetic alginate coating (Alg-Mag-CHMs) to offer a minimal distance and easy separation. The Alg-Mag-CHMs fabrication was achieved using layer-by-layer encapsulation. The thickness could be tailored by applying different numbers of coating layers. Alg-Mag-CHMs had a denser alginate hydrogel shell, which prevented the risk of cell leakage. After mixture, Alg-Mag-CHMs could be rapidly separated in magnet field and the coating could be easily washed off. This study presented proof of concept data of a non-contact 3D coculture model that offered many useful features.
1.
Introduction
Coculture models that have long been used to study the interactions between cell populations are fundamental to cell-cell interaction investigations[1-3]. They provide the culture condition for growing two types of cells together, either by direct contact or non-contact models. In recent years, the coculture models had been increasingly popular in tissue engineering. The synergetic interactions between cell populations could facilitate the extracellular matrix (ECM) production, despite the success would be depended on the distance between cells[4,5]. The direct contact coculture would provide a better bioactive molecule exchange to facilitate the cell-cell interactions, but the applications were limited to cells from autologous source. Allogeneic or xenogeneic cells can be obtained easily but cannot be implanted due to immunological reactions. The most popular non-contact coculture model were based on transwell technique, as a two-dimensional (2D) cell culture[6,7]. Many researchers had modified it for 3D coculture, such as the hydrogel coculture[8]. However, the distance between target cells and signaling cells in these models is still relatively long. Moreover, the possible cell leakage from hydrogels represented a risk. In this study, we proposed a conceptual non-contact 3D coculture model based on collagen hydrogel microsphere (CHM) with removable superparamagnetic alginate coating (Alg-Mag-CHMs). The removable superparamagnetic alginate coating was added onto the surface of CHM via layer-by-layer assembly. Alg-Mag-CHMs could be rapidly separated by an external magnetic field from mixture. 2.
Materials and Methods
2.1 MSCs isolation and culture. Neonatal New Zealand rabbit (Breeding Farm for Sichuan Provincial Experimental Animal Special Committee) was sacrificed. Bone marrow was flushed out with culture medium
(alpha-modified Eagle’s medium (α-MEM, Sigma) containing 20% fetal bovine serum (FBS, GIBCO), penicillin 100 U/mL and streptomycin 100 g/mL). Cells were cultured at 37°C with 5% CO2. Non-adherent cells were removed by replacing medium after 24 h. 2.2 Fabrication of collagen hydrogel microspheres (CHMs). CHMs were made from collagen type I (extracted from new born calf skin). Collagen solution was neutralised with 1M NaOH and adjusted to the final concentration of 6.5 mg/mL with the 6
cell solution. The final cell density was 5.0×10 cells/mL. The cell-collagen mixture was injected into the stirring (500 rpm) polydimethylsiloxane for 30 min at 4C and then kept stirring for another 20 min at 37C. After gelation, CHMs were washed with -MEM. 2.3 Encapsulation of CHMs with superparamagnetic alginate coating. Coating solution was prepared with alginate solution (3% w/v, Sigma-Aldrich) and superparamagnetic beads (2mg/mL, 1 m, K&B Sphere Tech). CHMs were immersed in the 102 mM CaCl2 solution for 10 sec and then transferred into the coating solution for 10 sec, before washed with normal saline solution. Above procedure was used to get one-layer of coating, and multiple coating layers could be achieved by repeating the coating procedures. CHMs with alginate coating was defined as Alg-CHM and CHM with superaramagnetic coating was named as Alg-Mag-CHM. 2.4 Characterization of coated CHMs. Coating thickness. CHM and Alg-CHM were imaged by inverted microscope (Leica, DMI4000B). CHMs with fluorescent red latex bead (1 m, Sigma-Aldrich) embedded was also fabricated to visually distinguish the CHM and its coating layer. Coating thickness was measured by software (NanoMeasurer 1.2).
Surface Morphology. Samples were fixed in 0.25% glutaraldehyde at 4°C. After lyophilisation all samples were observed with scanning electron microscopy (SEM, Hitachi S-4800). Cytotoxicity test. Encapsulated CHMs were cultured for 24h before fluorescence staining. Samples were washed and immersed into fluorescein diacetate (FDA, 1 g/mL, Solarbio) and propidium iodide (PI, 1 g/mL, Solarbio) for 5 min and imaged by confocal laser scanning microscopy (CLSM, TCS SP 5, Leica). The surface of culture dish was also imaged by microscope at day 3 to observe the leakage of cells. 2.5 Separation of Alg-Mag-CHMs The separation of Alg-Mag-CHMs was validated with a mixture of CHMs and Alg-Mag-CHMs (1:1) in a transparent glass tube. To visually distinguish the two CHM samples, Alg-Mag-CHMs were stained by Toluidine Blue (TB) dye to mark the alginate layer in purple colour. Two CHMs were well-mixed and then closed to a magnet to finish the separation. 2.6 Removal of coating The alginate coating can be dissolved using sodium citrate solution, while preserving the cell viability[9]. Alg-Mag-CHMs was added with 5 mL sodium citrate (SC) solution (55 mM) to remove the coating and then observed under microscope. 3.
Results and Discussion
The CHMs size was fabricated between 200 m to 500 m, which had been found to enhance the mass transfer [10]. The coating process of CHM was shown in Figure 1A. The photos demonstrated the appearances of fluorescent red marked CHMs without coating, with one-layer coating, and with six-layer coating. Alginate coating appeared to be semi-transparent and covered the CHM completely. The encapsulation thickness was visually increased and quantitated from ~10 to ~120 m with one to
six coating layers (Figure 1B). Since the Alg-Mag layer represented the only barrier between two microspheres when CHMs and Alg-Mag-CHMs were in a close contact, the encapsulation thickness would be theoretically the minimal distance between two microspheres in mixture. As one coating, the minimal distance between two microspheres would be ~10 m, which would favor mass transfer in non-contact culture. The distance could be further tailored by the coating thickness and dispersion density, which would potentially provide the flexibility to measure more cell-cell signaling, such as juxtacrine, paracrine, and endocrine at different distances[11]. Figure 1C demonstrated the surface morphologies of CHM and Alg-Mag-CHM with two coating layers. After coating, visible collagen fibers in CHM were covered by the dense alginate networks in Alg-Mag-CHM. The different surface morphologies could impact cell behavior.
Figure 1. A) CHM with fluorescent red latex bead without coating, with one-layer, and six-layer coating. Scale bar: 100 m. B) Encapsulation thickness. C) Surface morphologies of the CHM and Alg-Mag-CHM. Scale bar: 500 m, 10 m and 1 m.
Figure 2A showed the viability of cell before and after coating. The live cells were stained in green and no obvious dead cell (red) can be observed, which indicated the coating process had no cytotoxicity. It also demonstrated that the coating would not obstruct nutrient supply. After 3 days’ culture,
spreading cells were observed on the surface of culture dish, suggesting cell leakage from CHMs (Figure 2B). In contrast, no leaking cell was observed from Alg-Mag-CHM. The alginate coating could prevent cells migrating out because there were less cell adhesion sites in alginate than collagen [12]. It would be potentially a critical feature in coculture, as the cell leakage may cause risk of allogeneic or xenogeneic cells invasion.
Figure 2. A) Live/dead staining of cells in CHM and Alg-CHM before and after coating. Scale bar: 100 m. B) cell leakage in CHM and Alg-Mag-CHM after 24h in vitro culture, Scale bar: 500 m.
The TB stained Alg-Mag-CHMs and CHMs were well-mixed in glass tube (Figure 3A, left panel). The TB stained Alg-Mag-CHMs were separated within one second after moving towards the magnet while CHMs (semi-transparent) were unaffected (Figure 3A, right panel and supplementary video 1). Calcium works as a physical crosslinker between guluronic acid blocks in alginate chain and can be captured by SC. Therefore, calcium alginate hydrogel can be dissolved in the SC solution fast. After SC solution treatment, the purple staining on Alg-Mag-CHMs disappeared and turned back to semi-transparent (Figure 3B). Under a light microscope, the samples were semi-transparent, while Alg-Mag-CHMs were dark-black (Figure 3C). The removability of coating would also provide an useful feature for potential applications, especially when using the cultured tissues for in vivo implantation. Figure 3D showed that a group of Alg-Mag-CHMs could be immobilized at a fixed location by magnet (supplementary
video 2). It would potentially a useful feature for customized cell culture conditions or magnet targeted cell delivery applications.
Figure 3. A) CHM and Alg-Mag-CHM before and after separation. Purple arrows: marked Alg-Mag-CHM. White arrow: CHM. B) TB staining before and after the removal of coating. C) Alg-Mag-CHM before and after removal of superparamagnetic alginate coating. Scale bar: 100 m. D) Immobilization of Alg-Mag-CHMs at a fixed location.
4.
Conclusions
This work reported a conceptual non-contact 3D coculture model based on core-shell structured Alg-Mag-CHMs. It offered many useful features, such as a controllable micron-sized distance between two microspheres, prevention of cell leakage, no cytotoxicity, fast separation from sample mixture, and easy removal of superparamagnetic alginate coating. Acknowledgments This work was supported by National Key Research Program of China (2018YFC1105902 and 2016YFC1103202), Natural Science Foundation of China (31130021 and 51403134), Fundamental Research Funds for the Central Universities (YJ201747), Sichuan Science and Technology Program (2018RZ0039), and the 111 Project (No. B16033). Reference: [1]
E.A. Gosselin, R. Gomes, T. Torregrosa, A.C. Mendelsohn, J.L. Funderburgh, D.L. Kaplan, Multi‐layered silk film coculture system for human corneal epithelial and stromal stem cells, J Tissue Eng Regen Med. (2018) 285–295.
[2]
R. Krencik, K. Seo, J.V. Van Asperen, N. Basu, C. Cvetkovic, S. Barlas, R. Chen, C. Ludwig, C. Wang, M.E. Ward, L. Gan, P.J. Horner, D.H. Rowitch, E.M. Ullian, Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes, Stem Cell Reports. 9 (2017) 1745–1753.
[3]
P.M.J. Ahmed A. Arzouni, Andreia Vargas‐Seymour, Nance Nardi, Aileen J.F. King, Using Mesenchymal Stromal Cells in Islet Transplantation, Stem Cells Transl. Med. (2018) 559–563.
[4]
B.B. Nguyen, R.A. Moriarty, T. Kamalitdinov, J.M. Etheridge, J.P. Fisher, Collagen hydrogel scaffold promotes mesenchymal stem cell and endothelial cell coculture for bone tissue engineering, J Biomed Mater Res Part A. (2017) 1123–1131.
[5]
M. Ansorge, T. Pompe, Systems for localized release to mimic paracrine cell communication in vitro, J. Control. Release. 278 (2018) 24–36.
[6]
D.A.N. Zhao, C. Xue, S. Lin, S. Shi, Q. Li, M. Liu, X. Cai, Y. Lin, Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co-Culture Model, J. Cell. Physiol. (2016) 1548–1558.
[7]
J.D. Erndt-marino, M.S. Hahn, Probing the response of human osteoblasts following exposure to sympathetic neuron-like PC-12 cells in a 3D coculture model, J Biomed Mater Res Part A. (2017) 984–990.
[8]
P. Diaz-rodriguez, J.D. Erndt-marino, A Three-Dimensional Chondrocyte-Macrophage Coculture, Tissue Eng. Part A. 23 (2017) 101–114.
[9]
W. Leong, T.T. Lau, D.A. Wang, A temperature-cured dissolvable gelatin microsphere-based cell carrier for chondrocyte delivery in a hydrogel scaffolding system, Acta Biomater. 9 (2013) 6459–6467.
[10]
J. Liu, C. Yu, Y. Chen, H. Cai, H. Lin, Y. Sun, J. Liang, Q. Wang, Y. Fan, X. Zhang, Fast fabrication of stable cartilage-like tissue using collagen hydrogel microsphere culture, J. Mater. Chem. B. 5 (2017) 9130–9140.
[11]
L.Q. Timothy Quang Vu, Ricardo Miguel Bessa de Castro, Bridging the gap: microfluidic devices for short and long distance cell–cell communication, Lab Chip. (2017) 1009–1023.
[12]
A. Moshaverinia, X. Xu, C. Chen, K. Akiyama, M.L. Snead, S. Shi, Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration, Acta Biomater. 9 (2013) 9343–9350.
Conflict of interest
The authors have no conflict of interest.
A
core-shell
structured
collagen
hydrogel
microsphere
with
removable
superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China *Corresponding author: Qiguang Wang email: [email protected]
Graphical Abstract We proposed a conceptual 3D coculture model based on collagen hydrogel microsphere (CHM) with removable superparamagnetic alginate coating (Alg-Mag-CHMs). The removable coating was added onto the surface of micro-sized CHM via layer-by-layer assembly technique. After culture, Alg-Mag-CHMs could be rapidly separated in the magnetic field and the coating could be removed in mild conditions.
A
core-shell
structured
collagen
hydrogel
microsphere
with
removable
superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China *Corresponding author: Qiguang Wang email: [email protected]
Highlights
Superparamagnetic alginate (Alg-Mag) coated collagen microspheres for 3D cell culture.
The thickness of Alg-Mag coating is tailorable with different number of layers.
The Alg-Mag coating is non-cytotoxic and prevents inner cell leakage.
The Alg-Mag microspheres can be rapidly separated from a mixture by magnetic field.
The Alg-Mag coating can be easily removed to retrieve the inner sample.
S0167-577X(19)30611-1 https://doi.org/10.1016/j.matlet.2019.04.059 MLBLUE 26042
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
23 January 2019 11 April 2019 14 April 2019
Please cite this article as: J. Liu, Q. Wang, Y. Sun, H. Lin, J. Liang, Y. Fan, X. Zhang, A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation, Materials Letters (2019), doi: https://doi.org/10.1016/j.matlet.2019.04.059
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A core-shell structured collagen hydrogel microsphere with removable superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China Corresponding author: * Qiguang Wang
email: [email protected]
Keywords Composite materials, Multilayer structure, Particles Abstract Coculture models have long been used in cell and tissue engineering studies. The direct contact model normally offers excellent cell-cell interactions, but difficult to separate the two cell types after coculture. The non-contact model offers a good cell separation, but the long cell-cell distance limited the cell interactions. This study proposed a conceptual 3D coculture model based on collagen hydrogel microspheres (CHMs) with removable superparamagnetic alginate coating (Alg-Mag-CHMs) to offer a minimal distance and easy separation. The Alg-Mag-CHMs fabrication was achieved using layer-by-layer encapsulation. The thickness could be tailored by applying different numbers of coating layers. Alg-Mag-CHMs had a denser alginate hydrogel shell, which prevented the risk of cell leakage. After mixture, Alg-Mag-CHMs could be rapidly separated in magnet field and the coating could be easily washed off. This study presented proof of concept data of a non-contact 3D coculture model that offered many useful features.
1.
Introduction
Coculture models that have long been used to study the interactions between cell populations are fundamental to cell-cell interaction investigations[1-3]. They provide the culture condition for growing two types of cells together, either by direct contact or non-contact models. In recent years, the coculture models had been increasingly popular in tissue engineering. The synergetic interactions between cell populations could facilitate the extracellular matrix (ECM) production, despite the success would be depended on the distance between cells[4,5]. The direct contact coculture would provide a better bioactive molecule exchange to facilitate the cell-cell interactions, but the applications were limited to cells from autologous source. Allogeneic or xenogeneic cells can be obtained easily but cannot be implanted due to immunological reactions. The most popular non-contact coculture model were based on transwell technique, as a two-dimensional (2D) cell culture[6,7]. Many researchers had modified it for 3D coculture, such as the hydrogel coculture[8]. However, the distance between target cells and signaling cells in these models is still relatively long. Moreover, the possible cell leakage from hydrogels represented a risk. In this study, we proposed a conceptual non-contact 3D coculture model based on collagen hydrogel microsphere (CHM) with removable superparamagnetic alginate coating (Alg-Mag-CHMs). The removable superparamagnetic alginate coating was added onto the surface of CHM via layer-by-layer assembly. Alg-Mag-CHMs could be rapidly separated by an external magnetic field from mixture. 2.
Materials and Methods
2.1 MSCs isolation and culture. Neonatal New Zealand rabbit (Breeding Farm for Sichuan Provincial Experimental Animal Special Committee) was sacrificed. Bone marrow was flushed out with culture medium
(alpha-modified Eagle’s medium (α-MEM, Sigma) containing 20% fetal bovine serum (FBS, GIBCO), penicillin 100 U/mL and streptomycin 100 g/mL). Cells were cultured at 37°C with 5% CO2. Non-adherent cells were removed by replacing medium after 24 h. 2.2 Fabrication of collagen hydrogel microspheres (CHMs). CHMs were made from collagen type I (extracted from new born calf skin). Collagen solution was neutralised with 1M NaOH and adjusted to the final concentration of 6.5 mg/mL with the 6
cell solution. The final cell density was 5.0×10 cells/mL. The cell-collagen mixture was injected into the stirring (500 rpm) polydimethylsiloxane for 30 min at 4C and then kept stirring for another 20 min at 37C. After gelation, CHMs were washed with -MEM. 2.3 Encapsulation of CHMs with superparamagnetic alginate coating. Coating solution was prepared with alginate solution (3% w/v, Sigma-Aldrich) and superparamagnetic beads (2mg/mL, 1 m, K&B Sphere Tech). CHMs were immersed in the 102 mM CaCl2 solution for 10 sec and then transferred into the coating solution for 10 sec, before washed with normal saline solution. Above procedure was used to get one-layer of coating, and multiple coating layers could be achieved by repeating the coating procedures. CHMs with alginate coating was defined as Alg-CHM and CHM with superaramagnetic coating was named as Alg-Mag-CHM. 2.4 Characterization of coated CHMs. Coating thickness. CHM and Alg-CHM were imaged by inverted microscope (Leica, DMI4000B). CHMs with fluorescent red latex bead (1 m, Sigma-Aldrich) embedded was also fabricated to visually distinguish the CHM and its coating layer. Coating thickness was measured by software (NanoMeasurer 1.2).
Surface Morphology. Samples were fixed in 0.25% glutaraldehyde at 4°C. After lyophilisation all samples were observed with scanning electron microscopy (SEM, Hitachi S-4800). Cytotoxicity test. Encapsulated CHMs were cultured for 24h before fluorescence staining. Samples were washed and immersed into fluorescein diacetate (FDA, 1 g/mL, Solarbio) and propidium iodide (PI, 1 g/mL, Solarbio) for 5 min and imaged by confocal laser scanning microscopy (CLSM, TCS SP 5, Leica). The surface of culture dish was also imaged by microscope at day 3 to observe the leakage of cells. 2.5 Separation of Alg-Mag-CHMs The separation of Alg-Mag-CHMs was validated with a mixture of CHMs and Alg-Mag-CHMs (1:1) in a transparent glass tube. To visually distinguish the two CHM samples, Alg-Mag-CHMs were stained by Toluidine Blue (TB) dye to mark the alginate layer in purple colour. Two CHMs were well-mixed and then closed to a magnet to finish the separation. 2.6 Removal of coating The alginate coating can be dissolved using sodium citrate solution, while preserving the cell viability[9]. Alg-Mag-CHMs was added with 5 mL sodium citrate (SC) solution (55 mM) to remove the coating and then observed under microscope. 3.
Results and Discussion
The CHMs size was fabricated between 200 m to 500 m, which had been found to enhance the mass transfer [10]. The coating process of CHM was shown in Figure 1A. The photos demonstrated the appearances of fluorescent red marked CHMs without coating, with one-layer coating, and with six-layer coating. Alginate coating appeared to be semi-transparent and covered the CHM completely. The encapsulation thickness was visually increased and quantitated from ~10 to ~120 m with one to
six coating layers (Figure 1B). Since the Alg-Mag layer represented the only barrier between two microspheres when CHMs and Alg-Mag-CHMs were in a close contact, the encapsulation thickness would be theoretically the minimal distance between two microspheres in mixture. As one coating, the minimal distance between two microspheres would be ~10 m, which would favor mass transfer in non-contact culture. The distance could be further tailored by the coating thickness and dispersion density, which would potentially provide the flexibility to measure more cell-cell signaling, such as juxtacrine, paracrine, and endocrine at different distances[11]. Figure 1C demonstrated the surface morphologies of CHM and Alg-Mag-CHM with two coating layers. After coating, visible collagen fibers in CHM were covered by the dense alginate networks in Alg-Mag-CHM. The different surface morphologies could impact cell behavior.
Figure 1. A) CHM with fluorescent red latex bead without coating, with one-layer, and six-layer coating. Scale bar: 100 m. B) Encapsulation thickness. C) Surface morphologies of the CHM and Alg-Mag-CHM. Scale bar: 500 m, 10 m and 1 m.
Figure 2A showed the viability of cell before and after coating. The live cells were stained in green and no obvious dead cell (red) can be observed, which indicated the coating process had no cytotoxicity. It also demonstrated that the coating would not obstruct nutrient supply. After 3 days’ culture,
spreading cells were observed on the surface of culture dish, suggesting cell leakage from CHMs (Figure 2B). In contrast, no leaking cell was observed from Alg-Mag-CHM. The alginate coating could prevent cells migrating out because there were less cell adhesion sites in alginate than collagen [12]. It would be potentially a critical feature in coculture, as the cell leakage may cause risk of allogeneic or xenogeneic cells invasion.
Figure 2. A) Live/dead staining of cells in CHM and Alg-CHM before and after coating. Scale bar: 100 m. B) cell leakage in CHM and Alg-Mag-CHM after 24h in vitro culture, Scale bar: 500 m.
The TB stained Alg-Mag-CHMs and CHMs were well-mixed in glass tube (Figure 3A, left panel). The TB stained Alg-Mag-CHMs were separated within one second after moving towards the magnet while CHMs (semi-transparent) were unaffected (Figure 3A, right panel and supplementary video 1). Calcium works as a physical crosslinker between guluronic acid blocks in alginate chain and can be captured by SC. Therefore, calcium alginate hydrogel can be dissolved in the SC solution fast. After SC solution treatment, the purple staining on Alg-Mag-CHMs disappeared and turned back to semi-transparent (Figure 3B). Under a light microscope, the samples were semi-transparent, while Alg-Mag-CHMs were dark-black (Figure 3C). The removability of coating would also provide an useful feature for potential applications, especially when using the cultured tissues for in vivo implantation. Figure 3D showed that a group of Alg-Mag-CHMs could be immobilized at a fixed location by magnet (supplementary
video 2). It would potentially a useful feature for customized cell culture conditions or magnet targeted cell delivery applications.
Figure 3. A) CHM and Alg-Mag-CHM before and after separation. Purple arrows: marked Alg-Mag-CHM. White arrow: CHM. B) TB staining before and after the removal of coating. C) Alg-Mag-CHM before and after removal of superparamagnetic alginate coating. Scale bar: 100 m. D) Immobilization of Alg-Mag-CHMs at a fixed location.
4.
Conclusions
This work reported a conceptual non-contact 3D coculture model based on core-shell structured Alg-Mag-CHMs. It offered many useful features, such as a controllable micron-sized distance between two microspheres, prevention of cell leakage, no cytotoxicity, fast separation from sample mixture, and easy removal of superparamagnetic alginate coating. Acknowledgments This work was supported by National Key Research Program of China (2018YFC1105902 and 2016YFC1103202), Natural Science Foundation of China (31130021 and 51403134), Fundamental Research Funds for the Central Universities (YJ201747), Sichuan Science and Technology Program (2018RZ0039), and the 111 Project (No. B16033). Reference: [1]
E.A. Gosselin, R. Gomes, T. Torregrosa, A.C. Mendelsohn, J.L. Funderburgh, D.L. Kaplan, Multi‐layered silk film coculture system for human corneal epithelial and stromal stem cells, J Tissue Eng Regen Med. (2018) 285–295.
[2]
R. Krencik, K. Seo, J.V. Van Asperen, N. Basu, C. Cvetkovic, S. Barlas, R. Chen, C. Ludwig, C. Wang, M.E. Ward, L. Gan, P.J. Horner, D.H. Rowitch, E.M. Ullian, Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes, Stem Cell Reports. 9 (2017) 1745–1753.
[3]
P.M.J. Ahmed A. Arzouni, Andreia Vargas‐Seymour, Nance Nardi, Aileen J.F. King, Using Mesenchymal Stromal Cells in Islet Transplantation, Stem Cells Transl. Med. (2018) 559–563.
[4]
B.B. Nguyen, R.A. Moriarty, T. Kamalitdinov, J.M. Etheridge, J.P. Fisher, Collagen hydrogel scaffold promotes mesenchymal stem cell and endothelial cell coculture for bone tissue engineering, J Biomed Mater Res Part A. (2017) 1123–1131.
[5]
M. Ansorge, T. Pompe, Systems for localized release to mimic paracrine cell communication in vitro, J. Control. Release. 278 (2018) 24–36.
[6]
D.A.N. Zhao, C. Xue, S. Lin, S. Shi, Q. Li, M. Liu, X. Cai, Y. Lin, Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co-Culture Model, J. Cell. Physiol. (2016) 1548–1558.
[7]
J.D. Erndt-marino, M.S. Hahn, Probing the response of human osteoblasts following exposure to sympathetic neuron-like PC-12 cells in a 3D coculture model, J Biomed Mater Res Part A. (2017) 984–990.
[8]
P. Diaz-rodriguez, J.D. Erndt-marino, A Three-Dimensional Chondrocyte-Macrophage Coculture, Tissue Eng. Part A. 23 (2017) 101–114.
[9]
W. Leong, T.T. Lau, D.A. Wang, A temperature-cured dissolvable gelatin microsphere-based cell carrier for chondrocyte delivery in a hydrogel scaffolding system, Acta Biomater. 9 (2013) 6459–6467.
[10]
J. Liu, C. Yu, Y. Chen, H. Cai, H. Lin, Y. Sun, J. Liang, Q. Wang, Y. Fan, X. Zhang, Fast fabrication of stable cartilage-like tissue using collagen hydrogel microsphere culture, J. Mater. Chem. B. 5 (2017) 9130–9140.
[11]
L.Q. Timothy Quang Vu, Ricardo Miguel Bessa de Castro, Bridging the gap: microfluidic devices for short and long distance cell–cell communication, Lab Chip. (2017) 1009–1023.
[12]
A. Moshaverinia, X. Xu, C. Chen, K. Akiyama, M.L. Snead, S. Shi, Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration, Acta Biomater. 9 (2013) 9343–9350.
Conflict of interest
The authors have no conflict of interest.
A
core-shell
structured
collagen
hydrogel
microsphere
with
removable
superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China *Corresponding author: Qiguang Wang email: [email protected]
Graphical Abstract We proposed a conceptual 3D coculture model based on collagen hydrogel microsphere (CHM) with removable superparamagnetic alginate coating (Alg-Mag-CHMs). The removable coating was added onto the surface of micro-sized CHM via layer-by-layer assembly technique. After culture, Alg-Mag-CHMs could be rapidly separated in the magnetic field and the coating could be removed in mild conditions.
A
core-shell
structured
collagen
hydrogel
microsphere
with
removable
superparamagnetic alginate coating for cell coculture and rapid separation Jun Liu, Qiguang Wang*, Yong Sun, Hai Lin, Jie Liang, Yujiang Fan, Xingdong Zhang National Engineering Research Center for Biomaterials, Sichuan University, Wangjiang Road 29, Chengdu 610064, China *Corresponding author: Qiguang Wang email: [email protected]
Highlights
Superparamagnetic alginate (Alg-Mag) coated collagen microspheres for 3D cell culture.
The thickness of Alg-Mag coating is tailorable with different number of layers.
The Alg-Mag coating is non-cytotoxic and prevents inner cell leakage.
The Alg-Mag microspheres can be rapidly separated from a mixture by magnetic field.
The Alg-Mag coating can be easily removed to retrieve the inner sample.