Date of Award
Doctor of Philosophy (PhD)
Sherif M Karam
Yasir E Griesh
In the stomach, epithelial stem cells are responsible for glandular homeostasis and continuous production of four main cell lineages secreting mucus, acid, pepsinogen and hormones. While alteration in the proliferation and differentiation program of these stem cells linked to the origin of gastric cancer, they represent an effective target for chemotherapy and a source for cell therapy or tissue engineering in cases of gastric mucosal damage or loss. The aim of this study were to 1) to manufacture various form of scaffolds using a biodegradable polymer (polycaprolactone), 2) to test the suitability of these scaffolds for growth of mouse gastric system (mGS) cells, and 3) to evaluate whether this culture system could sustain exposure to acidic environment for possible future applications.
Three form of polycaprolactone scaffold were fabricated: nonporous, microporous and microfibrous. Scanning electron microscopy (SEM) and mechanical testing revealed some similarities between the microfibrous scaffold and extra cellular matrix of mouse stomach wall. Examination of mGS cells seeded on different form of scaffold for 3 days using SEM and calcein viability assay revealed their preferential growth on microfibrous scaffolds fabricated by electrospinning technique.
Analysis of the growth pattern of mGS cells on microfibrous scaffolds following 3, 6, 9 and 12 days of culture using SEM and DNA PicoGreen assay demonstrated an initial increase in cell number, followed by reduction by days 9 and 12. To test whether this reduction was associated with cell differentiation, cryosections of cultured mGS cells on scaffolds were probed with gastric epithelial cell differentiation markers. On day 3, none of the markers bound to the cells. However, by day 9, approximately, 50% of the cells bound to N-acetyl-D-glucosamine-specific lectin (Griffonia simplicifolia II) suggesting differentiation into gland mucous cells. This finding was confirmed by the expression of trefoil factor 2 using immunocytochemistry. In addition, gene expression analysis using quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the expression of transcription factor SPDEF, required for differentiation of mucous cells, was gradually up-regulated with culture of mGS cells from 3 to 12 days.
To test whether this 3D culture system could tolerate the acidic environment of the stomach, the mechanical/chemical integrity of microfibrous scaffolds and cultured mGS cells were studied at acidic pH (3.0 to 7.4) using tensile strength measurements, fourier transform infrared spectroscopy, calcein assay, and mRNA/protein expression analysis. The in vitro wound-healing assay was also used to examine effects of acidic pH on cell migration. RPMI culture media at pH 3.0 and 4.5 reduced the mechanical integrity of scaffolds and significantly inhibited cell viability by > 70%. However, at pH 5.5 and 6.0, no significant change in cell viability and scaffold integrity was observed, but cell migration was inhibited by more than 50%. Interestingly, only after 3-day culture at pH 5.5, N-acetyl-D-glucosamine-specific lectin binding combined with significant up-regulation in the expression of SPDEF gene confirmed mucous cell differentiation.
In conclusion, a 3D culture model of mGS cells using microfibrous PCL scaffold supporting their differentiation into gland mucous cells has been established. Reducing the pH value of culture media to 5.5 modulates proliferation/migration programs of mGS cells and speeds up their differentiation into mucous cells. This study provides important basic information for the possible use of mGS cells and microfibrous PCL scaffolds for future gastric tissue engineering studies and regenerative therapy of some stomach diseases involving gastric mucosal damage or loss.
Pulikkot, Sunitha, "Establishment Of A 3d Culture Model of Gastric Stem Cells Supporting Their Differentiation into Mucous Cells Using Microfibrous Polycaprolactone Scaffold" (2015). Dissertations. 45.