Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Tissues and Ethics
2.2. Small Intestinal Crypt Isolation
2.3. HIO Maintenance
2.4. HIO Culture Medium
2.5. HIO Monolayer Culture
2.6. Immunofluorescence Staining of HIO Monolayers
2.7. Measurement of Paracellular Barrier Function of 3D HIO
2.8. Human PBMC Isolation and Culture
2.9. Flow Cytometry Analysis of Human PBMC
2.10. RNA Isolation and Quantitative Real-Time PCR
2.11. Microbiology Experiments
2.12. Whey Protein Isolate and Whey Protein Hydrolysates
2.13. Experimental Set-Up
2.14. Statistical Analyses
3. Results
3.1. Model Development
3.1.1. Differentiation from Crypt-Like to Villus-like HIO Monolayers
3.1.2. Effect of Hypoxia on Crypt-like HIO Monolayers and 3D HIO
3.1.3. Effect of Hypoxia on Villus-like HIO Monolayers
3.1.4. Differential Effect of Hypoxia on Crypt-like and Villus-like HIO Monolayers and 3D HIO
3.2. Comprehensive Screening of Whey Protein Fractions
3.2.1. Effect of WPI, DH28 and DH51 on Crypt-like HIO (Monolayer- and 3D-Cultured) in a Healthy Setting (Normoxia)
3.2.2. Effect of WPI, DH28 and DH51 on Villus-like HIO (Monolayer- and 3D-Cultured) in a Healthy Setting (Normoxia)
3.2.3. Effect of WPI, DH28 and DH51 on Crypt-like HIO (Monolayer- and 3D-Cultured) in a Diseased Setting (Hypoxia)
3.2.4. Effect of WPI, DH28 and DH51 on Villus-like HIO (Monolayer- and 3D-Cultured) in a Diseased Setting (Hypoxia)
3.2.5. Effect of WPI, DH28 and DH51 on T Cell Subsets and T Cell Proliferation
3.2.6. Effect of WPI, DH28 and DH51 on PBMC Cytokine Expression and Activation Makers
3.2.7. Effect of WPI, DH28 and DH51 on Four Microbial Strains
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primer | Forward | Reverse |
---|---|---|
β-actin | 5′-ATTGCCGACAGGATGCAGAAG-3′ | 5′-TTGCTGATCCACATCTGCTGG-3′ |
GAPDH | 5′-GGAAGCTCACTGGCATGGC-3′ | 5′-CCTGCTTCACCACCTTCTTG-3′ |
YWHAZ | 5′-TGAACTCCCCTGAGAAAGCC-3′ | 5′-TCCGATGTCCACAATGTCAAGT-3′ |
CD3e | 5′-TGCTGCTGGTTTACTACTGGA-3′ | 5′-GGATGGGCTCATAGTCTGGG-3′ |
IL8 | 5′-GCCGGAATACCTGGACTATGC-3′ | 5′-TTCCTTGGGGTCCAGACAGA-3′ |
OLFM4 | 5′-TGGACAGAGTGGAACGCTTG-3′ | 5′-TCAGAGCCACGATTTCTCGG-3′ |
LYS | 5′-GATAACATCGCTGATGCTGTAGCT-3′ | 5′-CATGCCACCCATGCTCTAATG-3′ |
IFABP | 5′-ACGGACAGACAATGGAAACGA-3′ | 5′-ACTGTGCGCCAAGAATAATGC-3′ |
MUC2 | 5′-CTACTGGTGTGAGTCCAAGG-3′ | 5′-GGCACTTGGAGGAATAAACTG-3′ |
PEPT1 | 5′-TGTCCACCGCCATCTACCATA-3′ | 5′-CCACGAGTCGGCGATAAGAG -3′ |
LAT2 | 5′-AGGCTGGAACTTTCTGAATTACG-3′ | 5′-ACATAAGCGACATTGGCAAAGA-3′ |
HIF1a | 5′-ATCCATGTGACCATGAGGAAATG-3′ | 5′-TCGGCTAGTTAGGGTACACTTC-3′ |
IL4 | 5′-AGTGTCCTTCTCATGGTGGC-3′ | 5′-CACCGAGTTGACCGTAACAG-3′ |
IL17 | 5′-CACTTTGCCTCCCAGATCAC-3′ | 5′-ACCAATCCCAAAAGGTCCTC-3′ |
IFNϒ | 5′-TGGCTTTTCAGCTCTGCATC-3′ | 5′-CCGCTACATCTGAATGACCTG-3′ |
TNFα | 5′-TCAATCGGCCCGACTATCTC-3′ | 5′-CAGGGCAATGATCCCAAAGT-3′ |
IL10 | 5′-TCCCTGTGAAAACAAGAGCA-3′ | 5′-ATAGAGTCGCCACCCTGATG-3′ |
Foxp3 | 5′- CACCTGGCTGGGAAAATGG-3′ | 5′-GGAGCCCTTGTCGGATGAT-3′ |
Strain | Culture Medium | Antibiotics Used as Growth Inhibition Control |
---|---|---|
Escherichia coli ATCC 25922 | BHI | colistin (2 μg/mL) |
Straphylococcus aureus ATCC 29213 | BHI | ampicillin (50 μg/mL) |
Lactobacillus rhamnosis | BHI | erythromycin (10 μg/mL) |
Bifidobacterium longum | MRS (+0.05% cystein) | erythromycin (10 μg/mL) |
Product | WPI | DH28 | DH51 |
---|---|---|---|
Protein (%) | 90.0 | 86.5 | 85.1 |
Lactose (%) | 0.05 | 0.10 | 0.09 |
Fat (%) | 0.10 | 0.10 | 0.07 |
Ash (%) | 4.0 | 3.3 | 4.9 |
Mn (Da) | N/A | 593 | 333 |
Mw (Da) | N/A | 914 | 581 |
<375 Da (%) | 16.1 | 37.3 | |
375–750 Da (%) | 35.9 | 36.4 | |
750–1250 Da (%) | 24.6 | 19.4 | |
1250–2500 Da (%) | 21.4 | 6.6 | |
>2500 Da (%) | 2.1 | 0.3 | |
DH (%) | 27.7 | 50.9 | |
FAA (%) | 0.0 | 0.5 | 29.0 |
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de Lange, I.H.; van Gorp, C.; Massy, K.R.I.; Kessels, L.; Kloosterboer, N.; Bjørnshave, A.; Stampe Ostenfeld, M.; Damoiseaux, J.G.M.C.; Derikx, J.P.M.; van Gemert, W.G.; et al. Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey. Nutrients 2023, 15, 393. https://doi.org/10.3390/nu15020393
de Lange IH, van Gorp C, Massy KRI, Kessels L, Kloosterboer N, Bjørnshave A, Stampe Ostenfeld M, Damoiseaux JGMC, Derikx JPM, van Gemert WG, et al. Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey. Nutrients. 2023; 15(2):393. https://doi.org/10.3390/nu15020393
Chicago/Turabian Stylede Lange, Ilse H., Charlotte van Gorp, Kimberly R. I. Massy, Lilian Kessels, Nico Kloosterboer, Ann Bjørnshave, Marie Stampe Ostenfeld, Jan G. M. C. Damoiseaux, Joep P. M. Derikx, Wim G. van Gemert, and et al. 2023. "Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey" Nutrients 15, no. 2: 393. https://doi.org/10.3390/nu15020393
APA Stylede Lange, I. H., van Gorp, C., Massy, K. R. I., Kessels, L., Kloosterboer, N., Bjørnshave, A., Stampe Ostenfeld, M., Damoiseaux, J. G. M. C., Derikx, J. P. M., van Gemert, W. G., & Wolfs, T. G. A. M. (2023). Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey. Nutrients, 15(2), 393. https://doi.org/10.3390/nu15020393