A xenogenic-free culture medium for cell micro-patterning systems as cell-instructive biomaterials for potential clinical applications

Cell micro-patterning controls cell fate and function and has potential for generating therapeutically usable mesenchymal stromal cell (MSC) populations with precise functions. However, to date, the micro-patterning of human cells in a translational context has been impossible because only ruminant media supplements, e.g., fetal bovine serum (FBS), are established for use with micro-patterns (MPs). Thus, there are currently no Good Manufacturing Practice (GMP)-compliant media available for micro patterns (MPs). This study-tested a xenogenic-free human plasma and platelet lysate (hP+PL) medium supplement to determine its compatibility with MPs. Unfiltered hP+PL medium resulted in significant protein deposition, creating a 'carpet-like' layer that rendered MPs ineffective. Filtration (3×/5×) eliminated this effect. Importantly, quantitative comparison using Droplet digital PCR revealed that human MSCs in all media types exhibited similar profiles with strong myogenic (CNN1/TAGLN2) and weaker osteogenic (ALPL/RUNX2) marker expression, and much weaker adipogenic (LPL/PPARG) and chondrogenic (COL2A1/ACAN) expression, with profiles being dominated by myogenic markers. Within these similar profiles, an even stronger induction of the myogenic marker TAGLN2 by all hP+PL- compared to FBS-containing media. Overall, this suggested that FBS can be replaced with hP+PL without altering differentiation profiles. However, assessing individual MSC responses to various MP types with defined categories revealed that unfiltered hP+PL medium was unusable. Importantly, FBS- and 3× filtered hP+PL media were comparable in each differentiation category. Summarized, this study recommends 3× filtered hP+PL as a xenogenic-free and potentially GMP compliant-alternative to FBS as a culture medium supplement for micro-patterning cell populations in both basic and translational research that will ensure consistent and reliable MSC micro-patterning for therapeutic use.&#xD.