Details
| Original language | English |
|---|---|
| Pages (from-to) | 57-71 |
| Number of pages | 15 |
| Journal | BIOMATERIALS |
| Volume | 171 |
| Early online date | 16 Apr 2018 |
| Publication status | Published - Jul 2018 |
Abstract
There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.
Keywords
- 3D bioprinting, Adipose stem cells, Cornea, Human collagen I, Human pluripotent stem cells, Laser-assisted bioprinting, Limbal epithelial stem cells, Recombinant human laminin, Extracellular Matrix/metabolism, Bioprinting, Humans, Ink, Cell Survival/drug effects, Collagen Type I/pharmacology, Lasers, Swine, Corneal Stroma/cytology, Cell Proliferation/drug effects, Cell Communication/drug effects, Limbus Corneae/cytology, Organ Culture Techniques, Human Embryonic Stem Cells/cytology, Tissue Engineering/methods, Cell Adhesion/drug effects, Epithelial Cells/cytology, Biomarkers/metabolism, Animals, Cornea/drug effects, Adipose Tissue/cytology, Printing, Three-Dimensional
ASJC Scopus subject areas
- Engineering(all)
- Mechanics of Materials
- Materials Science(all)
- Ceramics and Composites
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biophysics
- Materials Science(all)
- Biomaterials
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In: BIOMATERIALS, Vol. 171, 07.2018, p. 57-71.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks
AU - Sorkio, Anni
AU - Koch, Lothar
AU - Koivusalo, Laura
AU - Deiwick, Andrea
AU - Miettinen, Susanna
AU - Chichkov, Boris
AU - Skottman, Heli
N1 - Funding information: This study was financially supported by the Finnish Funding Agency for Innovation (Tekes) , the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital, Finland and the Academy of Finland . The authors further acknowledge financial support from Deutsche Forschungsgemeinschaft (DFG) , the Cluster of Excellence REBIRTH , and Biofabrication for NIFE project (Land Niedersachsen/Volkswagenstiftung). The authors alone are responsible for preparing the manuscript: the funders had no role in experiment planning, data collection and analysis as well as decision to publish. The authors also wish to thank Outi Melin, Hanna Pekkanen, Sari Kalliokoski, Anna-Maija Honkala and Emma Vikstedt for technical assistance and Pantec Engineering AG, (Ruggell, Liechtenstein) for supporting us with the Er:YAG Laser.
PY - 2018/7
Y1 - 2018/7
N2 - There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.
AB - There is a high demand for developing methods to produce more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α and p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.
KW - 3D bioprinting
KW - Adipose stem cells
KW - Cornea
KW - Human collagen I
KW - Human pluripotent stem cells
KW - Laser-assisted bioprinting
KW - Limbal epithelial stem cells
KW - Recombinant human laminin
KW - Extracellular Matrix/metabolism
KW - Bioprinting
KW - Humans
KW - Ink
KW - Cell Survival/drug effects
KW - Collagen Type I/pharmacology
KW - Lasers
KW - Swine
KW - Corneal Stroma/cytology
KW - Cell Proliferation/drug effects
KW - Cell Communication/drug effects
KW - Limbus Corneae/cytology
KW - Organ Culture Techniques
KW - Human Embryonic Stem Cells/cytology
KW - Tissue Engineering/methods
KW - Cell Adhesion/drug effects
KW - Epithelial Cells/cytology
KW - Biomarkers/metabolism
KW - Animals
KW - Cornea/drug effects
KW - Adipose Tissue/cytology
KW - Printing, Three-Dimensional
UR - http://www.scopus.com/inward/record.url?scp=85047424942&partnerID=8YFLogxK
U2 - 10.1016/j.biomaterials.2018.04.034
DO - 10.1016/j.biomaterials.2018.04.034
M3 - Article
C2 - 29684677
AN - SCOPUS:85047424942
VL - 171
SP - 57
EP - 71
JO - BIOMATERIALS
JF - BIOMATERIALS
SN - 0142-9612
ER -