Abstract
Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.
Highlights
Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP)
Non-GMP compliant 3T3 J2 feeders have been allowed in special clinical c ases[23], cells grown on a 3T3 feeder layer in foetal bovine serum (FBS) containing medium have
The cells were seeded into FBS-non-essential amino acid (NEAA) medium
Summary
Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells This method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet. The production of epithelial cell sheets for clinical use needs to be carried out under the regulatory and quality umbrella of good manufacturing practice (GMP) This is the minimum standard that a medicines manufacturer must meet in their production process, with specific regulations for human cell-based medicinal p roducts[5]. The concerns arising from the use of xenogeneic material in patients has led to research into alternatives for the growth of squamous epithelial cells, such as growth arrested dermal fibroblasts[25], MSC’s26 or omitting feeder layers all together[27]
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