Development of a Cellular-Based Assay for  Measuring the Anti-Inflammatory Potency of Donor Banked Milk

Marie-Lee Castonguay; Isabelle Pare; Steve J. Charette-; Lionel Loubaki

Development of a Cellular-Based Assay for Measuring the Anti-Inflammatory Potency of Donor Banked Milk

Marie-Lee Castonguay^1,2, Isabelle Pare¹, Steve J. Charette^2-4, Lionel Loubaki1,2*

¹Medical Affairs and Innovation, Héma-Québec, Quebec (QC), Canada

²Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec City, QC, Canada

³Institut de Biologie Intégrative et des Systèmes, Laval University; Quebec City, QC, Canada

⁴Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Quebec, Quebec City, QC, Canada

^*Corresponding author: Lionel Loubaki, Ph.D., Medical Affairs and Innovation, Héma-Québec, Quebec and Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Quebec City, QC, Canada

Received Date: 20 November 2022

Accepted Date: 24 November 2022

Published Date: 28 November 2022

Citation: Castonguay M-L, Pare I, Charette SJ, Loubaki L (2022) Development of a Cellular-Based Assay for Measuring the AntiInflammatory Potency of Donor Banked Milk. Arch Pediatr 7: 224. DOI: https://doi.org/10.29011/2575-825X.100224

Abstract

Background: Meeting extremely preterm infant nutritional needs are one of the main goals of the human milk feeding strategy in the treatment of necrotizing enterocolitis (NEC). Bioactive components of human milk also play an important role in the prevention of NEC. Thus, the objective of our study was to develop an assay to assess anti-inflammatory potential of specific milk samples (DHMs) through the monitoring of NFκB activation, a key factor that regulates inflammation. Methodology: Small intestine epithelial cells (FHs74) were seeded in 24-well plate and left to adhere for at least 18 hours after which the medium was removed and cells were incubated with DHMs or controls for 2h. Then, cells were washed with saline prior to the addition of peripheral blood mononuclear cell supernatant used as NFκB activator. After the stimulation, cells were recovered and the activation of NFκB was measured by flow cytometry or by ELISA. Results: Using this model, we were able to identify DHMs with strong, moderate and no anti-inflammatory ability. Variation among the triplicate in the same assay was less than 5% and variation between different assay performed at separate times was ≤10% supporting the reproducibility of this assay. Conclusion: Coupled to nutritional information, this potency assay could help breast milk banks or neonatal intensive care unit personnel improve their strategy during the selection and preparation of breast milk batches and favor a product with high clinical efficacy for the preterm infant.

Keywords: Breast milk; Potency assay; Clinical efficiency; Inflammation

Introduction

Cell-based assays and analysis are vital experimental tools in life science research and biomanufacturing. They are based on cell culture methods, where live cells are grown in vitro and used as model systems to assess the biochemistry and physiology of both healthy and diseased cells. Cell culture assays provide a means of quantitatively analyzing the presence, amount, or functional activity of a cell or tissue of interest [1].

Necrotizing enterocolitis (NEC) is a multifactorial disease affecting the gastrointestinal tract in 5 to 10% of preterm infants who weigh less than 1500 g at birth. The average mortality caused by NEC is between 20 and 30% and it can reach nearly 50% in infants requiring surgical management [2]. Despite a high volume of research in clinical as well as laboratory settings, we still possess only a limited understanding of the pathophysiological mechanisms of this devastating illness. Multiple etiologic factors including immaturity of the preterm newborn intestinal tract, formula feeding, infections, and ischemia have been associated with NEC. A combination of these risk factors, perhaps based on genetic predisposition, possibly lead to the mucosal and epithelial injury that is the initiating event of NEC [3, 4].

The intestinal epithelial lining made up of a single layer of multiple cell types that perform different functions including nutrient absorption, preventing entry of pathogenic organisms and unprocessed antigens, innate inflammatory signalling, secretion of molecules that contribute to the mucosal barrier, antigen presentation to underlying immune cells, and production of endocrine signalling molecules [3]. Many studies have shown how cytokines such as IFN-γ, TNF-α and IL-8, induced during intestinal inflammation can further breakdown the intestinal epithelial barrier by causing necrosis as well as inhibition of mucus production by intestinal epithelial cells [5-7].

Among the treatment options that may be considered to prevent NEC, one key strategy has been providing human milk (HM) feedings, both mother’s own milk (MOM) and donor human milk (DHM), to preterm infants [2,8]. Indeed, both MOM and DHM contain several bioactive molecules susceptible to promote intestinal barrier function and thus contribute to their ability to prevent NEC, such as antibodies, anti-inflammatory cytokines and growth factors [9]. However, there is currently no in-vitro test that can predict the anti-inflammatory potential of MOM and/or DHM.

The transcription factor nuclear factor-κB (NFκB) that consists of five subunits (p50, p65, p52, cRel and RelB) is a major regulator of inflammation by inducing the production of many cytokines, including IL-1, IL-6, chemokines and TNF-α and PAF all known to play an important role in inflammatory response during NEC [10, 11]. NFκB need to homo- or heterodimerize to form active NFκB [12, 13]. Among these, p50-p50 homodimers and p50-p65 heterodimers are the NFκB dimers mostly found in intestinal tissues [9, 10]. In addition, it has been reported that the expression of IκB, the natural repressor of NFκB, is decreased in immature enterocytes and that human breast milk could stimulate IκB expression and thus inhibit NFκB activation. This is a mechanism by which human breast milk could protect neonates from inflammatory bowel diseases such as NEC [16].

Herein, we report on the development of a potency assay to assess the ability of MOM and/or DHM to inhibit inflammation. This method based on the use of FHs74 intestinal cells and the monitoring of NFκB p65 phosphorylation. This method allows for standard evaluation of anti-inflammatory potential of MOM or DHM.

Materials and Methods

Human Donor Milk Collection

This study has been approved by Héma-Quebec’s research ethics committee and all participants in this study have signed an informed consent. Non-pasteurized human donor milk samples were provided by the Héma-Québec breast milk bank as well as by the regulatory analysis department of Héma-Québec. DHM was subjected to Holder pasteurization (62, 5˚C for 30 minutes in a water bath).

FHs74 Int cell expansion

The FHs74 Int cells were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA). These cells were extracted from the small intestine and thus are composed of a mixed population of epithelial cells including enterocytes, paneth cells and potentially some stem cells [17]. FHs74 Int cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Thermo Fisher Scientific, Waltham, MA, USA), at 37°C, 5% CO₂supplemented with 20% qualified fetal bovine serum (FBS) (Thermo Fisher Scientific), GlutaMAX 1X (Thermo Fischer Scientific), 10 µg/mL insulin (from bovine pancreas; Sigma Life Science) and 30 ng/mL of recombinant human epidermal growth factor (Thermo Fisher Scientific) to generate a master cell bank. Briefly, cells were seeded at the density of 4000 cells/cm² in T175 flasks for cell culture. Medium was changed after four days. At day seven of culture, cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS, Thermo Fisher) and harvested using TrypLE^TM Express (Thermo Fisher) for 10 minutes (min) at room temperature (RT). The cell suspension was diluted by adding an equal volume of Plasma-Lyte A (Baxter Canada, Mississauga, ON, Canada) containing 10% Human Serum Albumin (HSA) (Alburex^® 25, CSL Behring, Ottawa, ON, Canada). Cells were centrifuged (600 x g, 10 min), suspended in Plasma-Lyte A containing 5% HSA and the cell suspension was analyzed (count and viability) on Nucleocounter^® NC-250^™ (ChemoMetec, Lincoln, NE, USA). FHs74 cells were then reseeded as described above for one or two additional passages and kept frozen in liquid nitrogen vapor phase until use.

Preparation of Peripheral Blood Mononuclear Cell Supernatants

Human Peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation over Ficoll-Paque (GE Healthcare BioScience) from whole blood collected from healthy volunteers who signed an informed consent. To prepare the supernatants, PBMCs (5x10⁵ cells/well in a 24-well plate) were activated using 20 ng/mL of anti-human CD3 and CD28 antibodies (respectively clone OKT3 and CD28.2, both from Thermo Fisher). PBMCs were maintained with the antibody mixture at 37 °C, 5% CO₂ four days after which, supernatants were collected and frozen for further use. In addition, cells were also collected to ensure the activation state of the T-cells. Therefore, the latter were labelled with 10 µL of FcR block (Miltenyi Biotech, Auburn, USA), 4 µL of anti-CD3 (SK7 clone; Thermo Fisher) and 12, 3 µL of 7AAD (Thermo Fisher) before being analyzed using flow cytometry.

PBMCs were considered activated with an expansion factor ≥ 3.

Development of the in Vitro Inflammation Model

All assays were performed using FHs74 cells at passage four. Thus, upon thaw using Thawstar (BIOLIFE Solutions; Bothell, WA, USA) and centrifugation (130 x g, 7 minutes) cells were counted and suspended in IMDM supplemented with 1% FBS. 10⁵ cells were seeded onto 24-well plate and allow adhering for at least 18 hours at 37 °C, 5% CO₂.

The next day, culture media was removed and a new culture medium, DHM or milk formulation diluted 1/50 in IMDM (Enfamil A+ Gentlease, Mead Johnson Nutrition, Kanata, ON, CA) was added and incubated for 2 hours with the FHs74 cells according to the following experimental conditions: FH74s alone (baseline); FHs74 stimulated (positive control); FHs74 incubated with milk formulation (negative control); FHs74 incubated with the donor milk to be tested. After this first step, FHs74 cells were washed with DPBS before the addition of different pro-inflammatory factors such as PBMCs supernatants (200 µL; previously filtered with 0.22 µm filter), lipopolysaccharides (LPS) from Klebsiella pneumoniae (1 µg/mL); from Escherichia coli O55:B5 (1 µg/ mL); and from Pseudomonas aeruginosa (1 µg/mL; all LPS from Sigma), as well as IFN-