This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.
In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 µg/mL, 100 µg/mL, 200 µg/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ. The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.
Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in preterm infants1. The disease is characterized by severe intestinal inflammation that can rapidly deteriorate to intestinal necrosis, sepsis, and potentially death. Although the etiology is unclear, evidence suggests NEC is multifactorial and the result of a complex interaction of feeding, abnormal bacterial colonization, and an immature intestinal epithelium2,3. Preterm infants have increased intestinal permeability, abnormal bacterial colonization, and low enterocyte regenerative capacity4,5, increasing their risk for intestinal barrier dysfunction, bacterial translocation, and NEC development. Therefore, identifying strategies or interventions to accelerate intestinal epithelial maturation and promote regeneration or healing of the intestinal epithelium is critical in preventing this deadly disease.
Studies have demonstrated that human milk (HM) is protective against NEC in preterm infants6,7,8,9,10,11. Both human and animal studies have shown that bovine-based formula increases intestinal permeability and is directly toxic to intestinal epithelial cells2,12. Although not fully elucidated, evidence suggests the protective effects of HM are mediated through bioactive components such as lactoferrin, immunoglobulin A (IgA), and HM oligosaccharides13. HM is also rich in hyaluronan (HA), a uniquely nonsulfated glycosaminoglycan with repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharides14,15. Importantly, we have shown that oral 35 kDa HA (HA35), an HM HA mimic, attenuates the severity of intestinal injury, prevents bacterial translocation, and decreases mortality in a murine NEC-like intestinal injury model16,17.
Here, the effects of HA35 on intestinal healing and regeneration in vitro are further investigated. Currently, the most widely used in vitro assay for intestinal wounding and repair is a scratch wound assay performed in colorectal cancer (CRC) cell monolayers. The physiological relevance of such a model to the preterm infant intestine is limited, as wound repair of CRC cells relies heavily upon the highly proliferative nature of cancer cells rather than stem cell-driven repair processes18. To overcome this limitation, the establishment of a 2D enteroid scratch wound model, including the procedure of isolating and maintaining primary stem cell-derived small intestinal enteroids from preterm non-human primates (NHP), is described here. Given preterm NEC is most often reported in the distal small intestine, the use of primary epithelial cell organoids in a model of intestinal damage and repair provides a more physiologically translatable in vitro model compared with existing models utilizing traditional colorectal monolayers18,19.
All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. Following institutional approval, fetal small intestine convenience samples from a preterm non-human primate (NHP, 90% gestation, olive baboon, Papio anubis) were obtained following euthanasia for a separate study (Protocol #101523-16-039-I)20.
1. Establishment of preterm non-human primate 3D intestinal enteroids
2. Establishment of enteroid monolayer and scratch wound assay
The effects of HA on tissue repair and wound healing in various tissues and organs are well-documented; however, the specific effects of HA with a molecular weight of 35 kDa on fetal or neonatal small intestinal healing and regeneration are currently unknown. To test the ability of HA35 to promote wound healing in a model of the fetal or neonatal small intestine, we generated 3D intestinal enteroids from NHP ileal tissue and further dissociated this tissue into single cells to create 2D enteroid-derived monolayers (Figure 1A,B). The monolayers were grown to confluency and manually scratched using a P200 pipette tip (Figure 1B,C). Monolayers were then treated with HA35 (50 µg/mL, 100 µg/mL, 200 µg/mL) or control (PBS). Cell migration and proliferation into the gap were imaged every 4 h for a total of 24 h using a transmitted-light microscope equipped for live-cell imaging22. The rate of wound closure, a measure of the speed of cell migration, was quantified using ImageJ as percent wound healing using the Wound Healing Size Plugin21 (Figure 2). The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h (step 2.4.6.). As shown in Figure 3, exposure to HA35 resulted in a dose- and time-dependent stimulation of cell migration/proliferation, leading to accelerated wound closure. HA35 at 100 µg/mL and 200 µg/mL significantly increased healing by ~1.5-2.5-fold relative to control at both 4 h and 12 h (*p < 0.05).
Figure 1. Enteroid generation and enteroid-derived monolayer wound healing assay procedure. (A) Culture of three-dimensional non-human primate intestinal enteroids used to dissociate into (B) two-dimensional monolayers. (C) Monolayers were grown to >90% confluence in a 24-well plate and then treated with hyaluronic acid 35 kDa or phosphate-buffered saline for 24 h. Monolayers were then scratched with a P200 pipette tip. Scale bar = 800 µm. Please click here to view a larger version of this figure.
Figure 2. Wound healing assay images and analysis. Representative images of hyaluronic acid 35 kDa (100 µg/mL, 200 µg/mL) and control treatments of non-human primate enteroid monolayers. Images were obtained at 0 h, 4 h, 12 h, 16 h, and 24 h after performing a scratch with a P200 pipette tip. Images were taken at 4x magnification with the live-cell analysis instrument and analyzed in ImageJ using the Wound Healing Size Plugin. Percent wound healing was calculated from migrating/proliferating cells over 24 h. Scale bar = 800 µm. Please click here to view a larger version of this figure.
Figure 3. Effect of hyaluronic acid 35 kDa (HA35) on wound healing in enteroid monolayers. Change in wound healing over time in enteroid monolayers, dependent on HA35 treatment concentration. HA35 significantly increased wound healing after 4 h and 12 h at 100 µg/mL and 200 µg/mL when compared to control, but healing rates converged among treatments by 24 h. Significance was assessed using the Student's t-test at *p < 0.05, presented as mean ± standard error of the mean (SEM) (n = 6 wells per treatment). Please click here to view a larger version of this figure.
The gastrointestinal tract of a preterm infant is under continual regenerative pressure from repeated exposures to environmental insults associated with dysbiosis, inflammatory bacterial metabolites and toxins, and intermittent hypoxia23,24. Unfortunately, the intestinal epithelium of the preterm infant is unable to rapidly establish functional integrity23, resulting in barrier dysfunction, increased intestinal permeability, and, in severe cases, rampant intestinal inflammation and NEC development.
Glycosaminoglycans (GAGs) are a class of polysaccharides prevalent in human milk, which are potential bioactive factors protecting against the development of NEC15,16. HA is a linear polymer of repeating disaccharides of glucuronic acid and N-acetylglucosamine25,26 produced by hyaluronan synthases. HA can have either pro- or anti-inflammatory properties depending on the size and tissue environment27,28. In the intestine and colon, endogenous HA is present in the extracellular space adjacent to crypt epithelial cells and drives epithelial proliferation and normal intestinal growth29,30,31. HA is also a natural component in HM and is produced at the highest concentrations during the first weeks of lactation30. Notably, HA purified from HM or commercially available HA of molecular weight ~35 kDa (HA 35) protect against bacteria-induced colitis by increasing the expression of the antimicrobial β-defensin and the TJ protein ZO-1 in the colonic epithelium in vivo and vitro25,27. It is important to note that, among all the specific-sized HA (4.7 kDa, 16 kDa, 28 kDa, 74 kDa), HA35 is the most potent inducer of TJ proteins and antimicrobial peptide expression in the colonic epithelium in vitro. Moreover, large MW HA (~2,000 kDa) exerts no effect on TJ protein expression, indicating that these effects are size specific32. We also showed that oral administration of HA35 accelerates small intestinal maturation, promotes epithelial proliferation and differentiation, and protects against the development of NEC16. Here, the ability of HA35 to promote wound healing is tested using an in vitro scratch wound healing assay. Using the models described above, it was found that HA35 accelerates wound closure in a concentration-dependent manner at 4 h and 12 h post-scratch compared to control, clarifying a critical gap in knowledge on the effects of specifically sized HA on intestinal wound healing and tissue repair and, thus, confirming a beneficial role of HA35 in neonatal intestinal wound healing. Though the mechanism by which HA35 exerts this effect is unknown, the authors' previous data in mouse pups showed the mechanistic target of the rapamycin (mTOR) complex 1 (mTORC1) pathway was upregulated in ileal tissues after HA35 treatment16. Further studies are needed to determine the contribution of this pathway to these observed effects.
Multiple in vitro models have been utilized to investigate interventions promoting intestinal regeneration, healing, and repair. The most utilized in vitro assay for intestinal wounding and subsequent repair is the scratch wound assay, performed most commonly in colorectal cancer (CRC) cell monolayers33,34. The physiological relevance of such a model to the preterm infant intestine, however, is limited, as wound repair of CRC cells relies so heavily upon the highly proliferative nature of cancer cells rather than stem cell-driven repair processes18. The recent ability to isolate and maintain enteroids derived from crypts of intestinal epithelium provides an opportunity to explore a more physiologically relevant intestinal response to a multitude of immune or noxious stimuli, as well as possible therapeutic interventions35. Enteroids contain all the differentiated epithelial cell types found in the intestinal segment from which they are derived, thus serving as an effective model for studying intestinal barrier damage and repair36,37,38.
Described herein are the establishment and maintenance of an in vitro enteroid model of intestinal epithelial damage and repair using ileum from preterm NHP. This model allows for the investigation of mechanistic pathways and therapeutic interventions involved in epithelial healing and regeneration. A critical step in this protocol is the creation of a well-defined gap with a similar width, minimizing well-to-well variation. In addition, when scratching the monolayers, the pipette tip should maintain constant contact with the bottom of the well to cleanly remove the cell layer. Excessive pressure should be avoided to prevent "lifting" of the edges of the wound. Similarly, the post-wounding PBS wash step should be performed gently, using the sidewall of the well, to avoid damage to the remaining monolayer or additional widening of the wound gap. Finally, one must avoid keeping the plate below 37 °C for extended periods to ensure the ECM coating on the plate remains gel-like and the monolayer is not disrupted due to ECM viscosity changes.
Though this model has distinct advantages over traditional cell culture, it is technically more demanding, expensive, and time-consuming compared to the traditional wound healing assay in CRC monolayers. Furthermore, consistency in the reagents and media used is critical in ensuring the survival and proper maintenance of these cultures, as is maintaining the cells, reagents, and ECM at proper temperatures. Finally, primary small intestinal monolayers are notoriously short-lived, so the wound assay described here should be conducted soon after monolayer confluency is achieved.
Taken together, these results suggest that small intestinal enteroid monolayers can be used as a novel model to investigate intestinal epithelial regeneration through the processes of cell migration and proliferation following wounding. In addition, enteroids provide a much more physiologically translatable tissue to study effects on differentiated intestinal cell viability. Such models may provide a platform to dissect the mechanisms regulating epithelial repair and aid in the identification of novel therapeutic targets.
The authors have nothing to disclose.
This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. HC is supported by grant P20GM134973 from the National Institutes of Health. KB is supported by a Children's Hospital Foundation (CHF) and Presbyterian Health Foundation (PHF) grant. Live-cell imaging services provided by the Cancer Functional Genomics core were supported partly by the National Institute of General Medical Sciences Grant P20GM103639 and National Cancer Institute Grant P30CA225520 of the National Institutes of Health, awarded to the University of Oklahoma Health Sciences Center Stephenson Cancer Center.
10 mL Serological Pipet | Fisher Scientific | 13-675-49 | |
100x21mm Dish, Nunclon Delta | ThermoFisher Scientific | 172931 | |
15 mL Conical tube | VWR | 89039-666 | |
24-Well, TC-Treated, Flat Bottom Plate | Corning | 3524 | |
37 µM Reversible Cell Strainer | STEMCELL Technologies | 27215 | |
50 mL Conical tube | VWR | 89039-658 | |
70 µm Sterile Cell Strainers | Fisher Scientific | FB22-363-548 | |
Albumin, Bovine (BSA) | VWR | 0332-100G | |
CellTiter-Glo 3D Cell Viability Assay | Promega | G9681 | |
Dulbecco's Modified Eagle's Medium/Nutrient Ham's Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer | STEMCELL Technologies | 36254 | |
Gentle Cell Dissociation Reagent | STEMCELL Technologies | 100-0485 | |
ImageJ | NIH | imagej.nih.gov/ij/ | |
Incucyte S3 Live-Cell Analysis Instrument | Sartorius | 4647 | |
Incucyte Scratch Wound Analysis Software Module | Sartorius | 9600-0012 | |
IntestiCult Organoid Growth Medium (Human) | STEMCELL Technologies | 06010 | This is HOGMY, but without the Y-27632 or antibiotics. Also used as base for HOGM, but then only missing the antibiotics. |
Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography | Millipore Sigma | L3012-10MG | |
Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free | Corning | 356231 | |
Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate | ThermoFisher Scientific | 136101 | |
PBS (Phosphate-Buffered Saline), 1X [-] Calcium, Magnesium, pH 7.4 | Corning | 21-040-CM | |
Primocin | Invivogen | ant-pm-1 | This is broad-spectrum antibiotics |
Sodium Hyaluronate, Research Grade, HA20K | Lifecore Biomedical | HA20K-1 | |
TC20 Automated Cell Counter | Company: Bio-Rad | 1450102 | |
Trypsin-EDTA 1X, 0.25% Trypsin | Fisher Scientific | MT25053CI | |
Y-27632 | STEMCELL Technologies | 72302 |