A New Immortalized Human Lacrimal Gland Cell Line
Abstract
:1. Introduction
2. Materials and Methods
2.1. Surgical Removal and Patient Description Germany
2.2. Surgical Removal and RNA Sequencing from Lacrimal Glands
2.3. Electroporation
2.4. Tissue Preparation and Cell Culture
2.5. Collagen Coating
2.6. RNA Purification and Sequencing
2.7. RNA Sequencing and Data Analysis
2.8. cDNA Synthesis
2.9. Reverse Transcription (RT)-PCR and Gel Electrophoresis
2.10. Semiquantitative RT-PCR Analysis
2.11. Fluorescence Immunostaining of PAX6,FOXC1/2, CLDN5, KRT19 and OCLN
2.12. Fluorescence Immunostaining of SV40
2.13. Fluorescein Isothiocyanate (FITC)-Dextran Permeability Assay
2.14. Transepithelial Electrical Resistance (TEER) Measurement
2.15. Spheroid Culture
2.16. Histological Staining
2.17. Spheroid Analysis Area, Perimeter, Circularity, and Roundness
2.18. Three-Dimensional Culture in Extracellular Matrix Environment
2.19. Transmission Electron Microscopy
3. Results
3.1. Generation of Immortalized Lacrimal Gland Cell Lines
3.2. Total RNAseq of Immortalized Human Lacrimal Gland Cells
3.3. Validation of Lacrimal Gland Markers at the Transcriptional and Protein Levels
3.4. Utilization of Immortalized Cells in a 3D Spheroid Model
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence | Product Size (Base Pairs) | Annealing Temperature [°C] |
---|---|---|---|
ACTA2 | GCCGAGATCTCACCGACTAC TCACGGACAATCTCACGCTC | 88 | 60 |
ACTB | GATCCTCACCGAGCGCGGCTACA GCGGATGTCCACGTCACACTTCA | 298 | 60 |
AQP5 | TCCATTGGCCTGTCTGTCAC CTTTGATGATGGCCACACGC | 232 | 63 |
CADH1 | AGGGGTTAAGCACAACAGCA ACGACGTTAGCCTCGTTCTC | 118 | 60 |
CD34 | TAGACTGTGCAGTGATGTGGT GGCAGACTTGGCTAAAGGTCC | 105 | 60 |
CD90 | TGGATTAAGGATGAGGCCCG GGGGAGGTGCAGTCTGTATT | 116 | 60 |
CST6 | GGCAGCAACAGCATCTACTACTT ACAGTTGTGCTTTAGGAGCTGAG | 255 | 59 |
CSTB | CGTGTCATTCAAGAGCCAGG CGCTCTGGTAGACGGAGGAT | 243 | 59 |
FOXC1 | TCGGCTTGAACAACTCTCCA GACGTGCGGTACAGAGACTG | 82 | 60 |
hu18S | GGAGCCTGAGAAACGGCTA TCGGGAGTGGGTAATTTGC | 64 | 60 |
LTF | CAGACCGCAGACATGAAACT TTCAAGAATGGACGAAGTGT | 479 | 60 |
LYZ | CTCTCATTGTTCTGGGGC ACGGACAACCCTCTTTGC | 350 | 60 |
MYL9 | ACCCACCAGAAGCCAAGATG GCGTTGCGAATCACATCCTC | 334 | 63 |
PAX6 | TAACCTGCCTATGCAACCCC ATAACTCCGCCCATTCACCG | 91 | 60 |
PAX6 | AGTTCTTCGCAACCTGGCTA TGAACGTGCTGCTGATAGGA | 500 | 57 |
PIGR | AATGCTGACCTCCAAGTGCTAAAG ATCACCACACTGAATGAGCCATCC | 242 | 60 |
VIM | GCTTCAGAGAGAGGAAGCCG AAGGTCAAGACGTGCCAGAG | 82 | 60 |
VWF | AGAACAGATGTGTGGCCCTG CTTCCGGTCCTGACAGACAC | 113 | 60 |
Antibody | Host Species | Dilution | Company |
---|---|---|---|
PAX6 | Sheep | 1:50 | R&D Systems, Minneapolis, MN, USA |
FOXC1/2 | Rabbit | 1:500 | Cohesion, Suzhou, China |
SV40 | Mouse | 1:50 | Merck-Millipore, Darmstadt, Germany |
F-Actin | Mouse | 1:200 | Invitrogen, Waltham, MA, USA |
CLDN5 | Rabbit | 1:100 | Invitrogen, Waltham, MA, USA |
KRT19 | Mouse | 1:50 | DAKO, Santa Clara, CA, USA |
OCLN | Mouse | 1:250 | Thermo Fisher Scientific, Waltham, MA, USA |
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Gleixner, S.; Zahn, I.; Dietrich, J.; Singh, S.; Drobny, A.; Schneider, Y.; Schwendner, R.; Socher, E.; Blavet, N.; Bräuer, L.; et al. A New Immortalized Human Lacrimal Gland Cell Line. Cells 2024, 13, 622. https://doi.org/10.3390/cells13070622
Gleixner S, Zahn I, Dietrich J, Singh S, Drobny A, Schneider Y, Schwendner R, Socher E, Blavet N, Bräuer L, et al. A New Immortalized Human Lacrimal Gland Cell Line. Cells. 2024; 13(7):622. https://doi.org/10.3390/cells13070622
Chicago/Turabian StyleGleixner, Sophie, Ingrid Zahn, Jana Dietrich, Swati Singh, Alice Drobny, Yanni Schneider, Raphael Schwendner, Eileen Socher, Nicolas Blavet, Lars Bräuer, and et al. 2024. "A New Immortalized Human Lacrimal Gland Cell Line" Cells 13, no. 7: 622. https://doi.org/10.3390/cells13070622
APA StyleGleixner, S., Zahn, I., Dietrich, J., Singh, S., Drobny, A., Schneider, Y., Schwendner, R., Socher, E., Blavet, N., Bräuer, L., Gostian, A. -O., Balk, M., Schulze-Tanzil, G., Günther, C., Paulsen, F., & Arnold, P. (2024). A New Immortalized Human Lacrimal Gland Cell Line. Cells, 13(7), 622. https://doi.org/10.3390/cells13070622