An in vitro colon model, particularly one suited to high-throughput screening, has the potential to enhance understanding of cellular mechanisms and functions important in intestinal health and can be used for drug testing and drug permeation studies. While extensively studied, traditional monolayered cultures using immortalized colon cancer cell lines on transwell plates fail to accurately replicate the native intestinal epithelium’s complex architecture. To address this limitation, we have developed a novel, facile photopolymerization technique to fabricate scaffolds that closely resemble colon crypts. We have further developed a method using screen printing to be able to coat these scaffolds while preserving the crypt architecture in order to vary the surface chemistry of these systems. This article focuses on the development of 3D crypt models that can be made with simple equipment and with chemical precursors that are commercially available to make building tissue models more accessible to the broader research community.
DahlhamerJM, ZammittiEP, WardBW, et al.Prevalence of inflammatory bowel disease among adults aged >/=18 years - United States, 2015. MMWR Morb Mortal Wkly Rep, 2016; 65(42):1166–1169; doi: 10.15585/mmwr.mm6542a3
2.
NguyenGC, ChongCA, ChongRY. National estimates of the burden of inflammatory bowel disease among racial and ethnic groups in the United States. J Crohns Colitis, 2014; 8(4):288–295; doi: 10.1016/j.crohns.2013.09.001
3.
AxelradJE, LichtigerS, YajnikV. Inflammatory bowel disease and cancer: The role of inflammation, immunosuppression, and cancer treatment. World J Gastroenterol, 2016; 22(20):4794–4801; doi: 10.3748/wjg.v22.i20.4794
4.
BhattacharyaA, OstermanMT. Biologic therapy for ulcerative colitis. Gastroenterol Clin North Am, 2020; 49(4):717–729; doi: 10.1016/j.gtc.2020.08.002
5.
UyanikogluA, ErmisF, AkyuzF, et al.Infliximab in inflammatory bowel disease: Attention to adverse events. Eur Rev Med Pharmacol Sci, 2014; 18(16):2337–2342.
6.
NeurathMF. Current and emerging therapeutic targets for IBD. Nat Rev Gastroenterol Hepatol, 2017; 14(5):269–278; doi: 10.1038/nrgastro.2016.208
7.
SartorRB. Mechanisms of disease: Pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol, 2006; 3(7):390–407; doi: 10.1038/ncpgasthep0528
8.
HashashJG, FadelCGA, RimmaniHH, et al.Biologic monotherapy versus combination therapy with immunomodulators in the induction and maintenance of remission of Crohn’s disease and ulcerative colitis. Ann Gastroenterol, 2021; 34(5):612–624; doi: 10.20524/aog.2021.0645
9.
SheethalS, RatheeshM, JoseSP, et al.Anti-Ulcerative effect of curcumin-galactomannoside complex on acetic acid-induced experimental model by inhibiting inflammation and oxidative stress. Inflammation, 2020; 43(4):1411–1422; doi: 10.1007/s10753-020-01218-9
10.
GehartH, CleversH. Tales from the crypt: New insights into intestinal stem cells. Nat Rev Gastroenterol Hepatol, 2019; 16(1):19–34; doi: 10.1038/s41575-018-0081-y
11.
IsmailAS, HooperLV. Epithelial cells and their neighbors. IV. Bacterial contributions to intestinal epithelial barrier integrity. Am J Physiol Gastrointest Liver Physiol, 2005; 289(5):G779–G84; doi: 10.1152/ajpgi.00203.2005
12.
SnoeckV, GoddeerisB, CoxE. The role of enterocytes in the intestinal barrier function and antigen uptake. Microbes Infect, 2005; 7(7–8):997–1004; doi: 10.1016/j.micinf.2005.04.003
13.
BrandtzaegP. The gut as communicator between environment and host: Immunological consequences. Eur J Pharmacol, 2011; 668(Suppl 1):S16–S32; doi: 10.1016/j.ejphar.2011.07.006
14.
KleivelandCR. Co-cultivation of Caco-2 and HT-29MTX. In: The Impact of Food Bioactives on Health: in vitro and ex vivo models. (VerhoeckxK, CotterP, López-ExpósitoI, et al. eds.) Springer International Publishing: Cham; 2015; pp. 135–140.
15.
BekiarisV, PerssonEK, AgaceWW. Intestinal dendritic cells in the regulation of mucosal immunity. Immunol Rev, 2014; 260(1):86–101; doi: 10.1111/imr.12194
16.
MowatAM, AgaceWW. Regional specialization within the intestinal immune system. Nat Rev Immunol, 2014; 14(10):667–685; doi: 10.1038/nri3738
17.
WangY, AhmadAA, SimsCE, et al.In vitro generation of colonic epithelium from primary cells guided by microstructures. Lab Chip, 2014; 14(9):1622–1631; doi: 10.1039/c3lc51353j
MasaokaY, TanakaY, KataokaM, et al.Site of drug absorption after oral administration: Assessment of membrane permeability and luminal concentration of drugs in each segment of gastrointestinal tract. Eur J Pharm Sci, 2006; 29(3–4):240–250; doi: 10.1016/j.ejps.2006.06.004
20.
Lozoya-AgulloI, González-ÁlvarezI, González-ÁlvarezM, et al.In situ perfusion model in rat colon for drug absorption studies: Comparison with small intestine and Caco-2 cell model. J Pharm Sci, 2015; 104(9):3136–3145; doi: 10.1002/jps.24447
21.
TannergrenC, BergendalA, LennernäsH, et al.Toward an increased understanding of the barriers to colonic drug absorption in humans: Implications for early controlled release candidate assessment. Mol Pharm, 2009; 6(1):60–73; doi: 10.1021/mp800261a
22.
WalterE, JanichS, RoesslerBJ, et al.HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: In vitro-in vivo correlation with permeability data from rats and humans. J Pharm Sci, 1996; 85(10):1070–1076; doi: 10.1021/js960110x
23.
EschMB, SungJH, YangJ, et al.On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic ‘body-on-a-chip’ devices. Biomed Microdevices, 2012; 14(5):895–906; doi: 10.1007/s10544-012-9669-0
24.
BeduneauA, TempestaC, FimbelS, et al.A tunable Caco-2/HT29-MTX co-culture model mimicking variable permeabilities of the human intestine obtained by an original seeding procedure. Eur J Pharm Biopharm, 2014; 87(2):290–298; doi: 10.1016/j.ejpb.2014.03.017
25.
BurtonPS, ConradiRA, HoNF, et al.How structural features influence the biomembrane permeability of peptides. J Pharm Sci, 1996; 85(12):1336–1340; doi: 10.1021/js960067d
26.
BurtonPS, GoodwinJT, ConradiRA, et al.In vitro permeability of peptidomimetic drugs: The role of polarized efflux pathways as additional barriers to absorption. Adv Drug Deliv Rev, 1997; 23(1–3):143–156; doi: 10.1016/S0169-409X(96)00432-2
27.
Zweibaum A, Laburthe M, Grasset E, et al. Use of Cultured Cell Lines in Studies of Intestinal Cell Differentiation and Function. In: Comprehensive Physiology. 2011; pp. 223–255; doi: 10.1002/cphy.cp060407
28.
YuanH, ChenCY, ChaiGH, et al.Improved transport and absorption through gastrointestinal tract by PEGylated solid lipid nanoparticles. Mol Pharm, 2013; 10(5):1865–1873; doi: 10.1021/mp300649z
29.
CalatayudM, VazquezM, DevesaV, et al.In vitro study of intestinal transport of inorganic and methylated arsenic species by Caco-2/HT29-MTX cocultures. Chem Res Toxicol, 2012; 25(12):2654–2662; doi: 10.1021/tx300295n
30.
AllaireJM, CrowleySM, LawHT, et al.The intestinal epithelium: Central coordinator of mucosal immunity. Trends Immunol, 2018; 39(9):677–696; doi: 10.1016/j.it.2018.04.002
31.
HalmDR, HalmST. Secretagogue response of goblet cells and columnar cells in human colonic crypts. Am J Physiol Cell Physiol, 2000; 278(1):C212–C33; doi: 10.1152/ajpcell.2000.278.1.C212
32.
BiswasS, DavisH, IrshadS, et al.Microenvironmental control of stem cell fate in intestinal homeostasis and disease. J Pathol, 2015; 237(2):135–145; doi: 10.1002/path.4563
33.
FuchsE, ChenT. A matter of life and death: Self-renewal in stem cells. EMBO Rep, 2013; 14(1):39–48; doi: 10.1038/embor.2012.197
KosinskiC, LiVS, ChanAS, et al.Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl Acad Sci U S A, 2007; 104(39):15418–15423; doi: 10.1073/pnas.0707210104
36.
BrittanM, WrightNA. Stem cell in gastrointestinal structure and neoplastic development. Gut, 2004; 53(6):899–910; doi: 10.1136/gut.2003.025478
37.
HahnS, KimG, JinSM, et al.Protective effects of metformin in the pro-inflammatory cytokine induced intestinal organoids injury model. Biochem Biophys Res Commun, 2024; 690:149291; doi: 10.1016/j.bbrc.2023.149291
38.
Caipa GarciaAL, KucabJE, Al-SeroriH, et al.Tissue organoid cultures metabolize dietary carcinogens proficiently and are effective models for DNA adduct formation. Chem Res Toxicol, 2024; 37(2):234–247; doi: 10.1021/acs.chemrestox.3c00255
39.
SahooDK, MartinezMN, DaoK, et al.Canine intestinal organoids as a novel in vitro model of intestinal drug permeability: A proof-of-concept study. Cells, 2023; 12(9):1269; doi: 10.3390/cells12091269
40.
ChusilpS, LiB, LeeD, et al.Intestinal organoids in infants and children. Pediatr Surg Int, 2020; 36(1):1–10; doi: 10.1007/s00383-019-04581-3
41.
SatoT, StangeDE, FerranteM, et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology, 2011; 141(5):1762–1772; doi: 10.1053/j.gastro.2011.07.050
42.
MithalA, CapillaA, HeinzeD, et al.Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells. Nat Commun, 2020; 11(1):215; doi: 10.1038/s41467-019-13916-6
43.
OjoBA, VanDussenKL, RosenMJ. The promise of patient-derived colon organoids to model ulcerative colitis. Inflamm Bowel Dis, 2022; 28(2):299–308; doi: 10.1093/ibd/izab161
44.
CostaJ, AhluwaliaA. Advances and current challenges in intestinal in vitro model engineering: A digest. Front Bioeng Biotechnol, 2019; 7:144; doi: 10.3389/fbioe.2019.00144
45.
JungJP, BhuiyanDB, OgleBM. Solid organ fabrication: Comparison of decellularization to 3D bioprinting. Biomater Res, 2016; 20(1):27; doi: 10.1186/s40824-016-0074-2
46.
YueZ, LiuX, CoatesPT, et al.Advances in printing biomaterials and living cells: Implications for islet cell transplantation. Curr Opin Organ Transplant, 2016; 21(5):467–475; doi: 10.1097/mot.0000000000000346
47.
MacedoMH, TorrasN, García-DíazM, et al.The shape of our gut: Dissecting its impact on drug absorption in a 3D bioprinted intestinal model. Biomater Adv, 2023; 153:213564; doi: 10.1016/j.bioadv.2023.213564
48.
TorrasN, ZabaloJ, AbrilE, et al.A bioprinted 3D gut model with crypt-villus structures to mimic the intestinal epithelial-stromal microenvironment. Biomater Adv, 2023; 153:213534; doi: 10.1016/j.bioadv.2023.213534
49.
DubbinK, HoriY, LewisKK, et al.Dual-Stage crosslinking of a gel-phase bioink improves cell viability and homogeneity for 3D bioprinting. Adv Healthc Mater, 2016; 5(19):2488–2492; doi: 10.1002/adhm.201600636
50.
PandalaN, LaScolaMA, TangY, et al.Screen printing tissue models using chemically cross-linked hydrogel systems: A simple approach to efficiently make highly tunable matrices. ACS Biomater Sci Eng, 2021; 7(11):5007–5013; doi: 10.1021/acsbiomaterials.1c00902
51.
PandalaN, HaywoodS, LaScolaMA, et al.Screen printing to create 3D tissue models. ACS Appl Bio Mater, 2020; 3(11):8113–8120; doi: 10.1021/acsabm.0c01256
52.
HynesSR, RauchMF, BertramJP, et al.A library of tunable poly(ethylene glycol)/poly(L-lysine) hydrogels to investigate the material cues that influence neural stem cell differentiation. J Biomed Mater Res A, 2009; 89(2):499–509; doi: 10.1002/jbm.a.31987
53.
WangY, AhmadAA, ShahPK, et al.Capture and 3D culture of colonic crypts and colonoids in a microarray platform. Lab Chip, 2013; 13(23):4625–4634; doi: 10.1039/c3lc50813g
54.
Royce HynesS, McGregorLM, Ford RauchM, et al.Photopolymerized poly(ethylene glycol)/poly(L-lysine) hydrogels for the delivery of neural progenitor cells. J Biomater Sci Polym Ed, 2007; 18(8):1017–1030; doi: 10.1163/156856207781494368
55.
KwonO, LeeH, JungJ, et al.Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids. Nat Commun, 2024; 15(1):799; doi: 10.1038/s41467-024-45103-7
56.
DalloulF, MietnerJB, RaveendranD, et al.From unprintable peptidic gel to unstoppable: Transforming diphenylalanine peptide (Fmoc-FF) nanowires and cellulose nanofibrils into a high-performance biobased gel for 3D printing. ACS Appl Bio Mater, 2025; 8(3):2323–2339; doi: 10.1021/acsabm.4c01803
57.
DobosA, Van HoorickJ, SteigerW, et al.Thiol-Gelatin-Norbornene bioink for laser-based high-definition bioprinting. Adv Healthc Mater, 2020; 9(15):e1900752; doi: 10.1002/adhm.201900752
58.
JiangX, MietnerJB, HarderC, et al.3D printable hybrid gel made of polymer surface-modified cellulose nanofibrils prepared by surface-initiated controlled radical polymerization (SI-SET-LRP) and upconversion luminescent nanoparticles. ACS Appl Mater Interfaces, 2023; 15(4):5687–5700; doi: 10.1021/acsami.2c20775
59.
TsunekiM, HardeeS, MichaudM, et al.A hydrogel-endothelial cell implant mimics infantile hemangioma: Modulation by survivin and the Hippo pathway. Lab Invest, 2015; 95(7):765–780; doi: 10.1038/labinvest.2015.61
60.
LianP, BraberS, VarastehS, et al.Hypoxia and heat stress affect epithelial integrity in a Caco-2/HT-29 co-culture. Sci Rep, 2021; 11(1):13186; doi: 10.1038/s41598-021-92574-5
61.
ZhangL, ChengJ, ShenJ, et al.Ghrelin inhibits intestinal epithelial cell apoptosis through the unfolded protein response pathway in ulcerative colitis. Front Pharmacol, 2021; 12:661853; doi: 10.3389/fphar.2021.661853
62.
Lozoya-AgulloI, AraujoF, Gonzalez-AlvarezI, et al.Usefulness of Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji B coculture models to predict intestinal and colonic permeability compared to Caco-2 monoculture. Mol Pharm, 2017; 14(4):1264–1270; doi: 10.1021/acs.molpharmaceut.6b01165
63.
JungP, SatoT, Merlos-SuarezA, et al.Isolation and in vitro expansion of human colonic stem cells. Nat Med, 2011; 17(10):1225–1227; doi: 10.1038/nm.2470).
64.
TakahashiK, TanabeK, OhnukiM, et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007; 131(5):861–872; doi: 10.1016/j.cell.2007.11.019
65.
TakahashiK, YamanakaS. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006; 126(4):663–676; doi: 10.1016/j.cell.2006.07.024
66.
CrespoM, VilarE, TsaiSY, et al.Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing. Nat Med, 2017; 23(7):878–884; doi: 10.1038/nm.4355
67.
MithalA, HumeAJ, Lindstrom-VautrinJ, et al.Human pluripotent stem cell-derived intestinal organoids model SARS-CoV-2 infection revealing a common epithelial inflammatory response. Stem Cell Reports, 2021; 16(4):940–953; doi: 10.1016/j.stemcr.2021.02.019
68.
GleesonJP, EstradaHQ, YamashitaM, et al.Development of physiologically responsive human iPSC-Derived intestinal epithelium to study barrier dysfunction in IBD. Int J Mol Sci, 2020; 21(4):1438; doi: 10.3390/ijms21041438
69.
KondoS, MizunoS, HashitaT, et al.Establishment of a novel culture method for maintaining intestinal stem cells derived from human induced pluripotent stem cells. Biol Open, 2020; 9(1):bio049064; doi: 10.1242/bio.049064
70.
WattersDA, SmithAN, EastwoodMA, et al.Mechanical properties of the colon: Comparison of the features of the African and European colon in vitro. Gut, 1985; 26(4):384–392.
CiascaG, PapiM, MinelliE, et al.Changes in cellular mechanical properties during onset or progression of colorectal cancer. World J Gastroenterol, 2016; 22(32):7203–7214; doi: 10.3748/wjg.v22.i32.7203
73.
TzengSF, LavikEB. Photopolymerizable nanoarray hydrogels delivery CNTF and promote differentiation of neural stem cells. Soft Mater, 2010; 6(10)(:2208–2215.
74.
AndersonJM, Van ItallieCM, PetersonMD, et al.ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells. J Cell Biol, 1989; 109(3):1047–1056; doi: 10.1083/jcb.109.3.1047
75.
ItoS, OkudaS, AbeM, et al.Induced cortical tension restores functional junctions in adhesion-defective carcinoma cells. Nat Commun, 2017; 8(1):1834; doi: 10.1038/s41467-017-01945-y
76.
SpalingerMR, Sayoc-BecerraA, SantosAN, et al.PTPN2 regulates interactions between macrophages and intestinal epithelial cells to promote intestinal barrier function. Gastroenterology, 2020; 159(5):1763–1777.e14; doi: 10.1053/j.gastro.2020.07.004
77.
JelinskySA, DerksenM, BaumanE, et al.Molecular and functional characterization of human intestinal organoids and monolayers for modeling epithelial barrier. Inflamm Bowel Dis, 2023; 29(2):195–206; doi: 10.1093/ibd/izac212
78.
KimHJ, IngberDE. Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integr Biol (Camb), 2013; 5(9):1130–1140; doi: 10.1039/c3ib40126j
79.
KimHJ, LiH, CollinsJJ, et al.Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip. Proc Natl Acad Sci U S A, 2016; 113(1):E7–E15; doi: 10.1073/pnas.1522193112
80.
NobenM, VanhoveW, ArnautsK, et al.Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology. United European Gastroenterol J, 2017; 5(8):1073–1081; doi: 10.1177/2050640617722903
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
