Culture of human umbilical cord mesenchymal stromal cells in a three-dimensional human platelet lysate gel

Abstract

The traditional cell culture method after isolation from the body involves growing cells in 2 dimensions on plastic culture plate. However, the natural structure and physiology is 3 dimensions. To mimic in vivo environment, there has an increasing interest to find the way to maintain physiological properties. Here, we describe culturing human umbilical cord mesenchymal stromal cells (HUC-MSCs_in 3D setting using human platelet lysate gel. This gel is a fibrin-based structure like a blood clot. The preparation step of human platelet lysate (HPL) is by freeze- thaw cycles in order to release factors important for cells to grow and expand. Using of HPL to substitute for fetal bovine serum reduces potential cross contamination between species and xenogenicity. To maintain HPL media as a liquid, we add the anticoagulant heparin. Without adding anticoagulant, the gel will form. The aim of this study is to retrieve HUC-MSCs from HPL gel using Nattokinase, to characterize HUC-MSCs following the International Society for Cell Therapy’s MSC criteria, and to test a 3D invasion model with HPL-gel based structure. The result shows that using 1.75% Nattokinase at 60 minutes can recover the cells without reducing cell number and viability. After Nattokinase treatment, cells are able to attach to plastic and to increase in number. Moreover, they are able to differentiate into fat, bone, and cartilage no different from cells grown in 2D culture. However, to test surface markers by flow cytometry, all MSC markers are positive except CD 105. They are also positive of cell surface markers that should be negative. When seeded back to 2D culture for an additional passage, the MSCs meet ISCT criteria the same as control.