is considered to be pivotal for acceptable long-term patency rates of

small diameter vascular grafts [5, 6]. Especially for acellular vascular

grafts that exhibit on-site endothelialization after implantation, the

preclinical evaluation of their re-endothelialization potential is of

utmost importance to avoid graft failure related complications. But

while well-established small and large animal models for vascular

prostheses are already available for preclinical studies [7], further

establishment of in vitro methodology that targets to estimate

endothelialization processes for vascular grafts is inevitable, as evi-

denced by the Three Rs principles for the ethical use of animals for

scientific investigations [8]. For the evaluation of acellular grafts,

which are directly introduced into a complex physiological environ-

ment after implantation, dynamic cell culture models are more

plausible than the static cell culture models, because cultivation

under flow generated shear stress simulates in vivo conditions

resulting in a more functional endothelium [9]. To provide such

physiological stimulations, state of the art bioreactor systems are

available to study small caliber vascular grafts under perfusion and

pulsatile flow in vitro [10]. They usually consist of two chambers,

providing intraluminal pulsatile flow to provide culture medium

and shear stress for the endothelial cells, and a surrounding con-

tainer, that can be filled with smooth muscle cells and cell culture

medium, if necessary [11]. More complex bioreactor systems

include pressure and flow measurement, biosensors for surveillance

of nutrient content and metabolites in the medium and rotational

parts to overcome gravitational effects during the cultivation period

[12]. However, the whole complexity of the in vivo situation for

the aspired clinical application cannot be mimicked at once in

bioreactor setups in vitro. Especially if artificial biomaterials are

used for the scaffolds, additional questions, e.g., regarding the

initial cell adhesion need to be addressed in more simplified setups.

Accordingly, approaches like static or low flow seeding and

pre-cultivation techniques that were developed for the generation

of cellular grafts needs to be considered in preparative steps prior to

bioreactor-assisted cultivation [9–11]. The comparability between

the many different approaches existing today might therefore be

impacted and most importantly, the scientific question to be

answered might differ with regard to acellular scaffolds. Acellular

scaffolds are thought to be repopulated by host cells onsite after

implantation in vivo. Regarding the initial adherence, it should be

noted that even hours after seeding of the cells, only low flow rates

should be applied to prevent a washout of the seeded cells

[13]. But, the medium flow in the system should be high enough

to wash off non-adherent cells, while applying detectable shear

stress to the cells.

Here, we present a protocol to estimate the homing of endo-

thelial cells in artificial bacterial cellulose based acellular vascular

grafts and their physiological performance in a perfusion bioreactor

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Max Wacker et al.