Functional binding of human adipose-derived stromal cells: effects of extraction method and hypoxia pretreatment

Abstract

Human adipose-derived stromal cells (hASCs) were evaluated in vitro for their ability to bind vascular adhesion and extracellular matrix proteins to arrest (firmly adhere) under physiological flow conditions. hASCs were flowed through a parallel plate flow chamber containing substrates presenting immobilized type I collagen, fibronectin, E-selectin, L-selectin, P-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1) under static and laminar flow conditions (wall shear stress = 1 dyn/cm). hASCs were able to firmly adhere to type I collagen, fibronectin, VCAM-1, and ICAM-1 substrates, but not to any of the selectins. Pretreatment with hypoxia increased the ability of hASCs isolated by liposuction to adhere to VCAM-1 and ICAM-1, but this effect was not seen in cells isolated by tissue excision. These results indicate that hASCs possess the ability to adhere key adhesion proteins, illustrate the importance of hASC harvest procedure, and suggest mechanisms for homing in a setting where interaction with inflamed or injured tissue is necessary.

FIGURE 1. Parallel plate flow chamber set-up

Both laminar flow and static assays are facilitated using this parallel plate flow chamber device though which cells are perfused and allowed to interact with the substrate.

FIGURE 2. hASCs functionally adhere to specific proteins

A) Static adhesion assay. Average percentage of hASCs initially in a field of view that remain firmly adhered to Type I Collagen (n=3, N=3), VCAM-1 (n=16, N=10), ICAM-1 (n=16, N=9), fibronectin (n=11, N=8), P-selectin (n=4, N=4), E-selectin (n=5, N=4), or L-selectin (n=5, N=4) after the introduction of flow (wall shear stress equal to 1 dyn/cm²). B) Laminar flow assay. Average number of cells firmly adhered per mm² to Collagen Type I (n=3, N=3), VCAM-1 (n=16, N=7), ICAM-1 (n=15, N=7), and fibronectin (n=12, N=7). (± S.D.; * = statistically different from Tween 20 control, p≤0.05).

FIGURE 3. Liposuction-derived hASCs show increased functional adhesion capabilities in response to 48 hours of hypoxic culture, while excision-derived hASCs do not

A) Micrograph showing hASCs grown under normal culture conditions labeled with Hypoxyprobe-1 for presence of pimonidazole adducts compared with (B) hypoxic culture control.

FIGURE 4. Increase in hASC functional adhesion after culture in hypoxic conditions is highly dependent on cell isolation procedure

A) Static adhesion assay. Average percentage of normal culture condition (n=5, N=2) and hypoxia-treated (n=7, N=2) liposuction-derived hASCs initially in a FOV that remained adherent to VCAM-1 after induction of flow (wall shear stress equal to 1 dyn/cm² initiated after six minutes). Data was pooled from two female donors of similar age (39 and 31) and similar BMI (23.2 and 20.8) who both received liposuction procedures. Conversely, hASCs isolated by excision procedures showed no statistically significant change in their ability to adhere VCAM-1 (normoxia: n=3, N=2) following 48 hours of hypoxic culture (hypoxia: n=4, N=2) (data not shown). (± S.D.; * = statistically different from normally cultured cells on same substrate, p≤0.05). B) Laminar flow assay. Firm adhesion of hypoxia-cultured and normally cultured liposuction-derived hASCs to VCAM-1 (n=4 for both normoxia and hypoxia, N=1) and ICAM-1 (n=4 for both normoxia and hypoxia, N=1). Data is from a 31-year-old female donor (BMI 20.8). The differential response to hypoxic culture may be partially dependent on the specific adhesion molecule: hASCs from the same donor did not significantly increase their ability to adhere fibronectin (n=2 for both normoxia and hypoxia, N=1) (data not shown). Conversely, hASCs isolated from a female 37-year-old female donor (BMI 25.8) by panniculectomy excision procedures exhibited no significant change in their ability to firmly adhere VCAM-1 (n=2 for both normoxia and hypoxia, N=1) or ICAM-1 (n=2 for both normoxia and hypoxia, N=1) following 48 hours hypoxic culture (data not shown). (± S.D.; * = statistically different from normally cultured cells on same substrate, p≤0.05).