A short-term plastic adherence incubation of the stromal vascular fraction leads to a predictable GMP-compliant cell-product.

Introduction: Mesenchymal stromal/stem cells (MSCs) derived from fat tissue are an encouraging tool for regenerative medicine. They share properties similar to the bone marrow-derived MSCs, but the amount of MSCs per gram of fat tissue is 500x higher. The fat tissue can easily be digested by collagenase, releasing a heterogeneous cell fraction called stromal vascular fraction (SVF) which contains a variable amount of stromal/stem cells. In Europe, cell products like the SVF derived from fat tissue are considered advanced therapy medicinal product (ATMPs). As a consequence, the manufacturing process has to be approved via GMP-compliant process validation. The problem of the process validation for SVF is the heterogeneity of this fraction. Methods: Here, we modified existing purification strategies by adding an additional plastic adherence incubation of maximal 20 hours after SVF isolation. The resulting cell fraction was characterized and compared to SVF as well as cultivated adipose-derived stromal/stem cells (ASCs) with respect to viability and cell yield, the expression of surface markers, differentiation potential and cytokine expression. Results: Short-term incubation significantly reduced the heterogeneity of the resulting cell fraction compared to SVF. The cells were able to differentiate into adipocytes, chondrocytes, and osteoblasts. More importantly, they expressed trophic proteins which have been previously associated with the beneficial effects of MSCs. Furthermore, GMP compliance of the production process described herein was acknowledged by the national regulatory agencies (DE_BB_01_GMP_2017_1018). Conclusion: Addition of a short purification-step after the SVF isolation is a cheap and fast strategy to isolate a homogeneous uncultivated GMP-compliant cell fraction of ASCs.

Modulation of tight junction structure and function by kinases and phosphatases targeting occludin.

Tight junctions (TJs) typically represent the most apical contacts in epithelial and endothelial cell layers where they play an essential role in the separation of extracellular or luminal spaces from underlying tissues in the body. Depending on the protein composition, TJs define the barrier characteristics and in addition maintain cell polarity. Two major families of integral membrane proteins form the typical TJ strand network, the tight junction-associated MARVEL protein (TAMP) family members occludin, tricellulin, and MarvelD3 as well as a specific set of claudins. Occludin was the first identified member of these tetraspanins and is now widely accepted as a regulator of TJ assembly and function. Therefore, occludin itself has to be tightly regulated. Phosphorylation of occludin appears to be of central importance in this context. Here we want to summarize current knowledge on the kinases and phosphatases directly modifying occludin, and their role in the regulation of TJ structure, function, and dynamics.