Mechanical conditioning of human mesenchymal stem cells for enhancing vascular regeneration

Abstract

Human mesenchymal stem cells (hMSCs) are an appealing cell type for therapeutic applications but remain limited by poor efficacy in clinical trials. Here, we describe a conditioning technique that enhances the vascular regenerative properties of hMSCs and increases their expression of endothelial cell and pericyte markers. We also describe an alginate gel encapsulation protocol for delivering the conditioned cells. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).¹.

Keywords: Biotechnology and Bioengineering; Cell Biology; Cell Culture; Flow Cytometry/Mass Cytometry; Health Sciences; Model Organisms; Stem Cells; Tissue Engineering.

Graphical abstract

Figure 1

Preparation of mesenchymal stem cell culture and stretch plates (A) hMSCs are cultured in T75 flasks until Passage 5. (B) Stretch plates are assembled in the sterile cell culture hood. (C) Stretch plate silicone membranes are coated with fibronectin. (D) Coated silicone membranes are washed, dried, and sterilized under UV light for 24 h. (E) MSCs are seeded on the 96 well stretch plates.

Figure 2

The custom-built high throughput biaxial oscillatory stretch system (HT-BOSS) (A) The completed 96 well stretch plates are attached to the HT-BOSS. (B) The coolant system and HT-BOSS are turned on. (C) The brachial waveform at 7.5% maximal strain is applied to the hMSCs at a frequency of 0.1 Hz.

Figure 3

Setup of flow cytometry hMSC phenotype analysis The filter configuration of flow cytometry system is adjusted to match fluorescent spectra and minimize spectral overlap (upper panel). The spectral profiles of fluorescent dyes (lower panel).

Figure 4

Overview of flow cytometry phenotype gating strategy The optimal gating strategy for identifying endothelial cell and pericyte markers of mechanically conditioned MSCs in Flowjo software.

Figure 5

Preparation of gel extruder and fabrication of alginate beads (A) Gel extruder parts are assembled in the sterile cell culture hood. (B) Mechanically conditioned MSCs are encapsulated in alginate gel beads.

Figure 6

The hindlimb ischemia surgical model and laser speckle imaging analysis (A) Preparation of surgical workplace and surgical tools. (B) The left hindlimb femoral artery is ligated without damaging adjacent veins and nerves. (C) Setup of laser speckle imaging device. (D) Representative laser speckle images of control and mechanically conditioned mice. (E) The laser speckle images are converted to greyscale and blood flow intensity is analyzed with Photoshop. (F) Representative quantification of relative ischemic to contralateral hindlimb blood perfusion for control and mechanically conditioned mice.