Human adipose stromal vascular cell delivery in a fibrin spray

Abstract

Background aims: Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system.

Methods: SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography.

Results: SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells.

Conclusions: Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Schematic of the LipiVage Device. The LipiVage device was designed to harvest small volumes of autologous adipose tissue for cosmetic surgical applications. The device aspirates fat at low vacuum into a sterile chamber. Once inside, the fat cells are concentrated by filtration. Fluids and free oil are drawn into a waste canister. Diagram courtesy of Genesis Biosystems

Figure 2

Stem/Progenitor content of freshly isolated adipose stromal vascular cells. A representative sample is shown. Statistics in analytical regions represent the percent positive (mean ± SEM) for four independent samples. The gating population for each histogram is shown in square brackets and comprises the denominator for percent calculations. From left to right, top to bottom: Nonhematopoietic (Non-Heme) cells are identified on events gated to remove autofluorescent cells, cell clusters, and events with

Figure 3

Composite schematic representation of Adipose Stromal Vascular Progenitor spray deposition. Panel A: Fibrinotherm reagent warmer and mixer. Cells and reagents are warmed to 37° C in this device. Panel B: The Tissomat spray module regulates nitrogen gas used to spray the cells suspended in reconstituted fibrinogen. The Duploject syringe system is in the foreground. Panel C: Cells mixed with fibrinogen sealer protein solution are sprayed onto 8-well chamber slides using the Duploject syringe system. Fibrinogen containing the cell suspension is mixed with thrombin and calcium as they enter the Duploject spray nozzle. Panel D: Light photomicrograph (40 x objective) taken 24 hours after cell/fibrin deposition. Darker areas are the fibrin substrate. Lighter areas represent areas where cells have coalesced within the fibrin, forming refractile clusters.

Figure 4

Growth and differentiation of spray-deposited adipose stromal vascular cells. After one week in culture in the presence of dexamethasone, cell colonies can be seen associated with fibrin strands. Cells continue to grow and at 3 weeks are semi-confluent and a proportion has spontaneously differentiated into adipocytes as determined by oil red-O staining.

Figure 5

Endothelial progenitor cell survival and differentiation after spray deposition. Replicate cultures of spray-on cells cultured for 3 weeks in the presence of EGM2 spontaneously form endothelial tubules. The tubules are microvascular-like in structure with CD31+, CD34+ vWF+ cells in intimate association with alpha smooth muscle actin+ cells. Endothelial tubules grew on a matrix of fibrin-associated stromal cells (isolated blue nuclei).