A Safe and Efficient Method to Retrieve Mesenchymal Stem Cells from Three-Dimensional Fibrin Gels

Abstract

Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.

Bone marrow-derived mesenchymal stem cells enhance angiogenesis via their α6β1 integrin receptor

“…To retrieve cells from 3D cultures, fibrin gels were degraded with nattokinase (Japan Bio Science Laboratory Co., Ltd., Osaka, Japan), a Bacillus -derived serine protease that is known for its potent fibrinolytic activity, as previously described [31]. Briefly, a fibrinolytic solution was prepared by dissolving 50 FU/mL (fibrin degradation unit) of nattokinase in PBS containing 1 mM EDTA (Fisher).…”

Characterization and Angiogenic Potential of Human Neonatal and Infant Thymus Mesenchymal Stromal Cells

“…After 48 hours, fibrin gels containing spheroids or cell monolayers were digested with nattokinase (50 fibrinolytic units per milliliter, NSK-SD; Japan Bio Science Laboratory Co., Ltd, Osaka, Japan, http://jbsl-net.com/ english/index.html) [9] prior to RNA isolation and qPCR. The effects of modifying the ratio of cell types in combination cell spheroids with thymus MSC/HUVEC ratios of 1:3, 1:1, and 3:1 on angiogenic gene expression were also investigated.…”

Towards Mimicking the Fetal Liver Niche: The Influence of Elasticity and Oxygen Tension on Hematopoietic Stem/Progenitor Cells Cultured in 3D Fibrin Hydrogels

“…Cells were harvested from the hydrogels by enzymatic digestion of the fibrin using nattokinase (NSK-SD; Japan Bio Science Laboratory, Osaka, Japan) dissolved at 50 fibrin degradation units per mL (FU/mL) in PBS containing 1 mM EDTA (Fisher Scientific, USA) [ 67 ]. Hydrogels were washed with PBS, and then, the nattokinase solution was added for the minimum amount of time to achieve hydrogel degradation, which was 15 and 30 min for the 0.6 and 1.8 mg/mL hydrogels, respectively.…”