A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids
Corresponding Author
Joao N. Ferreira
Faculty of Dentistry, Excellence Centre in Regenerative Dentistry, Chulalongkorn University, Bangkok, Thailand
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Correspondence
Joao Nuno Ferreira, Faculty of Dentistry, Chulalongkorn University, Chalermnavamarach 80 Bldg., 12th floor, 34 Henri-Dunant Rd, Pathumwan, Bangkok 10330, Thailand.
Email: [email protected]
Search for more papers by this authorRiasat Hasan
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorGanokon Urkasemsin
Faculty of Veterinary Science, Department of Preclinical and Applied Animal Science, Mahidol University, Nakhon Pathom, Thailand
Search for more papers by this authorKiaw K. Ng
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorChristabella Adine
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorSujatha Muthumariappan
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorGlauco R. Souza
University of Texas Health Sciences Center at Houston, Houston, TX, USA
Nano3D Biosciences Inc., Houston, TX, USA
Search for more papers by this authorCorresponding Author
Joao N. Ferreira
Faculty of Dentistry, Excellence Centre in Regenerative Dentistry, Chulalongkorn University, Bangkok, Thailand
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Correspondence
Joao Nuno Ferreira, Faculty of Dentistry, Chulalongkorn University, Chalermnavamarach 80 Bldg., 12th floor, 34 Henri-Dunant Rd, Pathumwan, Bangkok 10330, Thailand.
Email: [email protected]
Search for more papers by this authorRiasat Hasan
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorGanokon Urkasemsin
Faculty of Veterinary Science, Department of Preclinical and Applied Animal Science, Mahidol University, Nakhon Pathom, Thailand
Search for more papers by this authorKiaw K. Ng
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorChristabella Adine
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorSujatha Muthumariappan
Faculty of Dentistry, Discipline of Oral and Maxillofacial Surgery, National University of Singapore, Singapore, Singapore
Search for more papers by this authorGlauco R. Souza
University of Texas Health Sciences Center at Houston, Houston, TX, USA
Nano3D Biosciences Inc., Houston, TX, USA
Search for more papers by this authorAbstract
Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (β3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.
CONFLICTS OF INTEREST
The authors have no conflict of interest except for G. R. S. The University of Texas MD Anderson Cancer Center (UTMDACC) and Rice University, together with their researchers, have filed patents on the technology and intellectual property reported here. G. R. S. has equity in Nano3D Biosciences, Inc. UTMDACC and Rice University manage the terms of these arrangements in accordance with their established institutional conflict of interest policies.
Supporting Information
| Filename | Description |
|---|---|
| term2809-sup-0001-sf1.tifTIFF image, 3.8 MB |
Figure S1. Diameter size variation of 3D spheres through the 7-day differentiation stage in M3DL and Control cultures. Seeding density was 2 × 104 cells per well. N = 6-9. |
| term2809-sup-0002-sf2.tifTIFF image, 20.3 MB |
Figure S2. Validating the immunoreactivity of all primary antibodies used in this study against Sus Scrofa species. All tissue specimens were from freshly biopsied adult porcine submandibular glands. A. Hematoxylin and eosin staining of an adult porcine submandibular gland tissue section. Legend: white arrowheads represent myoepithelial cells either surrounding ducts (D), serous demilunes (SD), or adjacent to basement membrane (black arrowhead); M- mucous acini; S - serous acini. Scale bar: 100 μm; Mag.: 20X. B. Immunofluorescence imaging of cryosections of fresh pig SG tissues. All primary antibodies had satisfactory immuno-reactivity, except for MIST1 and CD31 which had poor immuno-reactivity against these pig tissues. Scale bar: 100 μm; Mag.: 20X. |
| term2809-sup-0003-sf3.tifTIFF image, 3.1 MB |
Figure S3. Transmission electron micrograph of an organoid developed by M3DL after SG epithelial differentiation. Lumenized-like areas (L), tight junctions (arrow heads) and exocytosis of secretory vesicles (S) can be visualized. Mag.: 60,000X. Scale bar: 100 nm. |
| term2809-sup-0004-sf4.tifTIFF image, 293.3 KB |
Figure S4. Trans-epithelial electrical resistance of organoids after SGDC differentiation in the M3DL system when compared with transwell filters without organoids (Control). |
| term2809-sup-0005-sf5.tifTIFF image, 618.4 KB |
Figure S5. Optimization of Carbachol concentration (0–1000 μM) by determining pig SGDC viability. Cell viability was measured with Real-time-Glo MT assay (Promega, USA) after 72h. Values are bioluminescence percentages relative to control cells cultured without Carbachol supplementation. Error bars represent SD. |
| term2809-sup-0006-Table_S1.pptxPowerPoint 2007 presentation , 50.8 KB |
Table S1. List of primary antibodies (conjugated and unconjugated) used for flow cytometry or immunofluorescence imaging. |
| term2809-sup-0007-Table_S2.pptxPowerPoint 2007 presentation , 48 KB |
Table S2. Oligonucleotide forward and reverse primary sequences optimized for mRNA in porcine SG tissues and used for qPCR in this study. |
| term2809-sup-0008-Table_S3.pptxPowerPoint 2007 presentation , 46.3 KB |
Table S3. Expression of surface markers in SGDC monolayer cultures by flow cytometry. Values represent total average ± SEM from 3 different samples. Statistical significance was determined by student t-test comparing the two passages. |
| term2809-sup-0009-Table_S4.pptxPowerPoint 2007 presentation , 48.5 KB |
Table S4. Messenger RNA expression in SGDC monolayer cultures by qPCR. Values are calculated as fold change normalized to the house keeping gene (S29) and relative to freshly dissected SG tissue. Values represent total average S.E.M. from 3 different samples. Gene regulation: “+” represents up-regulated, “-” represents down-regulated, “=” represents normo-regulated. Significance between passages was determined using a student t-test. ND: not detected. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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