The role of human fibronectin- or placenta basement membrane extract-based gels in favouring the formation of polarized salivary acinar-like structures

Abstract

Head and neck cancer patients treated with radiotherapy commonly experience hyposalivation and oral/tooth infections, leading to a reduced quality of life. Clinical management is currently unsatisfactory for dry mouth. Thus, there is a need for growing salivary fluid-secreting (acinar) cells for these patients. However, functionally-grown salivary acinar cells are cultured in Matrigel, a product that cannot be used clinically, owing to its source from a mouse sarcoma. Therefore, finding a gel suitable for clinical use and possessing properties similar to that of Matrigel would allow biopsied salivary cells to be expanded in vitro and transplanted into the mouths of xerostomic patients. This study tested gels made with human placenta basement membrane extract (BME) or fibronectin for the growth and differentiation of human salivary biopsies into acinar cells. We report here that, following expansion of primary human salivary gland epithelial cells (huSGs) in serum-free medium, using these gels (made from human proteins) allowed morphological and functional differentiation of salivary ductal cells into acinar-like cells. These (human) gels gave comparable results to Matrigel, such as differentiation into polarized acinar 3D units or monolayers with tight junction proteins (claudin-1, -2, -3) and exhibiting adequate transepithelial electrical resistance, acinar proteins (AQP5, α-amylase, mucin-1, NKCC1) and acinar adhesion-related cell markers (CD44, CD166). Ultrastructural, mRNA and protein analyses confirmed the formation of differentiated acinar polarized cells. The mitotic activity was highest with human placenta BME gel. This human culture model provided a reproducible approach to studying human salivary cell expansion and differentiation for tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Cell culture of differentiated human salivary epithelial cells in a serum‐free and scalable suspension system: The salivary functional units model

“…To date, however, the expansion of the enzymatically obtained primary acinar cells to a clinically useful mass and the maintenance of an acinar‐specific maturation state have been the two biggest technological hurdles to overcome in vitro. Thus, research efforts in the past decade have largely focused on identifying tissue‐resident stem cells that can self‐renew (Lombaert et al, ) and/or encapsulating cells in various matrix‐derived scaffolds, in which salivary epithelial cells spontaneously organize into spheroids with acinar‐like properties (Cantara et al, ; Lilliu et al, ; Maria, Liu, El‐Hakim, Zeitouni, & Tran, ; Maria, Zeitouni, Gologan, & Tran, ; Pradhan‐Bhatt et al, ; Soscia et al, ; Srinivasan et al, ).…”

“…Thus, research efforts in the past decade have largely focused on identifying tissue-resident stem cells that can self-renew (Lombaert et al, 2016) and/or encapsulating cells in various matrix-derived scaffolds, in which salivary epithelial cells spontaneously organize into spheroids with acinar-like properties (Cantara et al, 2012;Lilliu et al, 2016;Maria, Liu, El-Hakim, Zeitouni, & Tran, 2016;Maria, Zeitouni, Gologan, & Tran, 2011;Pradhan-Bhatt et al, 2013;Soscia et al, 2013;Srinivasan et al, 2016).…”

A magnetic three‐dimensional levitated primary cell culture system for the development of secretory salivary gland‐like organoids

“…Differentiated 3D organoids at Day 7 (in the M3DL system) were transferred into transwell polyester membrane filters (Corning) using a magnetic pen (Nano3D Biosciences Inc., TX, USA). Trans‐epithelial electrical resistance (TER) was measured in two separate organoids, using an EMD Millipore Millicell‐ERS2 Volt–Ohm Meter (Thermo Fisher Scientific) as previously described (Maria et al, ; Maria, Zeitouni, Gologan, & Tran, ). Traditionally, TER measurements are done on cells monolayers.…”

“…The rising of 3D cell culture systems addresses the limitations of two‐dimensional cell monolayers where rigid substrates/surfaces are present. Conversely, 3D cell cultures can mimic in vivo cell–cell and cell‐matrix interactions allowing the creation of organoids with complex networks of different cellular compartments (Maria, Liu, El‐Hakim, Zeitouni, & Tran, ; Pringle et al, ; Srinivasan et al, ). These organoids can also recapitulate in vivo morphological, physiologic, and pathological conditions in SG secretory organs (Shin et al, ; Su et al, ).…”

3D Culture Histology Cryosectioned Well Insert Technology Preserves the Structural Relationship between Cells and Biomaterials for Time‐Lapse Analysis of 3D Cultures

“…The characterization of these cells’ growth in complex soft biomaterials is of importance for salivary tissue engineering research. Other groups have attempted to engineer salivary structures in biomaterials, including hyaluronic acid, polyethylene glycol, Matrigel, collagen gels, placenta basement membrane extract, and fibronectin . To test the applicability of the 3D‐cryo well insert, we tested it in four soft biomaterials: Matrigel, GrowDex, Myogel, and a composite Myogel + GrowDex.…”