Development of in vitro infant intestinal models to assess responses to infant formulae

Early postnatal nutrition and intestinal barrier maturation are intimately linked, with the neonatal gut characterized by higher permeability than in adults. The aim of this thesis was to develop and validate in vitro models that mimic the infant intestinal barrier. Once developed, these models were employed to study epithelial responses to digested human milk and infant formulae (IF). The central hypothesis was that physiologically relevant, infant-like epithelial models would better capture the biological effects of infant food on nutrient absorption and barrier function than the conventional adult-like Caco-2/HT29-MTX co-culture. In chapter 2, Caco-2/HT29-MTX polarized monolayers were treated with the short-chain fatty acid salt sodium butyrate (NaB), previously reported to increase permeability without harm. High NaB concentrations (50–125 mM) reduced transepithelial electrical resistance (TEER) and increased paracellular permeability of Caco-2 monolayers in a dose-dependent manner under hyperosmotic conditions. NaB uptake occurred via monocarboxylate transporters (MCT-1, SMCT-1). However, NaB caused elevated apoptosis (Caspase-3 activity), stress responses (p21, HSP-70), and cytokine secretion (IL6, IL8, TNF). Therefore, NaB disturbed barrier integrity, and this disruption was not reversible after compound removal. Partial recovery following steroid treatment indicated that NaB-treated Caco-2/HT29-MTX monolayers may be more suitable for modeling gut inflammation than for representing the infant barrier. In chapter 3, the bile-derived sodium glycodeoxycholate (GDC) was evaluated in Caco-2/HT29-MTX monolayers. Treatment with 0.8 mM GDC decreased TEER and increased lactulose permeability 1.6-fold without inducing cytotoxicity or inflammation. GDC also reduced occludin (OCLN) localization at tight junctions, increased acidic mucins, and elevated extracellular alkaline phosphatase activity, consistent with neonatal mucosal features. Crucially, the GDC effect was transient: TEER recovered within 4 h of removal, supporting its use as a controllable, reversible infant-like gut barrier model. In chapter 4, the infant-like GDC model was then employed to compare gastrointestinal digestion and epithelial responses to human milk, dairy-, soya-, and amino acid-based IFs. Using infant-specific digestion conditions, peptide and amino acid transport were quantified in both infant- and adult-like Caco-2/HT29-MTX monolayers. In infant-like monolayers, human milk uniquely enhanced OCLN–actin co-localization (P < 0.05) and increased the diversity and basolateral abundance of bioactive peptides identified by UHPLC-HRMS/MS. The amino acid-based IF increased OCLN fluorescence intensity but reduced TEER in adult-like monolayers, indicating formulation-specific differences in epithelial compatibility. Across all digesta, peptide diversity and transport were significantly greater in infant-like than adult-like models (P < 0.05), with human milk promoting the richest basolateral peptide profile. Chapter 5 examined the intestinal porcine enterocyte cell line IPEC-J2 as an alternative to Caco-2/HT29-MTX. This cell line, isolated from the jejunum of an unsuckled neonatal piglet, differentiates into tightly adherent, columnar epithelial monolayers exhibiting apical–basal polarity and dense brush borders. However, standardized culture protocols are lacking. Four media were compared for their effects on TEER, permeability, and differentiation. Porcine serum (PS10) supported the most balanced epithelial phenotype, with intermediate TEER (~2400 Ω × cm²), low lactulose:mannitol permeability, strong zonula occludens-1 localization, and low fibroblast marker S100A4 expression. Following exposure to in vitro–digested IF, PS10-cultured IPEC-J2 monolayers exhibited increased TEER and enhanced basolateral amino acid transport, demonstrating suitability for dietary bioavailability studies in infant nutrition. In conclusion, this thesis establishes two in vitro models of the infant gut barrier: GDC-treated Caco-2/HT29-MTX and PS10-cultured IPEC-J2. GDC treatment provides a reproducible and reversible system to model transient infant gut permeability, while the IPEC-J2 line offers a stable, species-relevant platform. Together, these models advance mechanistic understanding of gut maturation and offer novel tools to design next-generation infant formulas that better emulate the functional benefits of human milk.

Grantor dc:publisher

University College Cork

Year dc:date.issued

2025

Author dc:creator

• Bietto, Francesca

Advisor dc:contributor.advisor

• Lucey, Alice

• Infant gut barrier
• Sodium glycodeoxycholate
• Sodium butyrate
• Caco-2
• HT29-MTX
• IPEC-J2
• INFOGEST
• Human milk
• Infant formula
• Peptide transport
• Tight junctions
• TEER
• Gastrointestinal digestion
• Occludin

Statement dc:rights

• © 2025, Francesca Bietto.

Licence dc:rights.uri

• https://creativecommons.org/licenses/by/4.0/

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10468/18850

OAI identifier oai:identifier

oai:cora.ucc.ie:10468/18850