Development of vascular inspired electrospun hollow fibre membranes for use in 3D tissue culture

Long a mainstay in deciphering the mechanisms orchestrating cell behaviour in health and disease, now marred by insufficient cell-cell and cell-extracellular matrix interactions, the physiological relevance of two-dimensional tissue culture has been called into question. Modern three-dimensional tissue culture has been developed to mimic the cellular niche, yet these constructs remain limited to 200 𝜇m in diameter or risk ischemic tissue damage. Utilising unconfined advective mass transfer perfusion bioreactors go some way in overcoming this size limitation. However, fluid shear forces in perfused media leave the cellular niche in these liable to disruption.
In vivo, blood vessels facilitate confined advective mass transfer, transporting dissolved gasses and nutrients throughout the body. Fulfilling this role in vitro, hollow fibre membranes (HFMs) have been used to support cell growth for, but not exclusively, erythroids, and endothelial cells (ECs). However, membranes used to date in these platforms are non-biodegradable, rigid, and monolithic, and, as such, are divorced in structure and nature from native tissues. In coronary artery bypass surgery, good procedural outcomes are associated with good morphological and mechanical similarities between the vessel transplanted and bypassed. To exhaustively mimic the physiological condition, HFMs used to support large three-dimensional tissue culture platforms should morphologically and mechanically mimic blood vessels of similar size.
From the meta-analysis of tissue engineered vascular grafts presented in this thesis, it was identified that electrospun polycaprolactone (PCL) and polydioxanone (PDO) conveyed mechanical properties akin to that of medium sized blood vessels. Additionally, it was noted that pyridine may be used as a controlled functional doping additive to modulate the properties of electrospun materials.
Given this state of affairs, it was hypothesised that HFMs may be fabricated continuously by electrospinning in a reproducible way to mimic the morphological and mechanical properties of native medium-sized blood vessels while additionally facilitating nutrient exchange for tissue culture. Furthermore, these HFMs may be used as a substrate for biomedical research, potentially for the production of biomaterials intended for therapeutic application.
In answer to these hypotheses, this thesis presents a novel method for the continuous production of electrospun HFMs. Utilising this novel method PCL and PDO were solvent blended for the first time to produce defect free micro-fibre meshes, with a mass ratio of PCL/PDO of 0.43. Utilising this polymer blend, polymer concentration and pyridine were used to alter the micro-fibre diameter between 1.03 and 1.75 μm. These meshes were demonstrated to produce membranes which had porosities which ranged between 88-92 %. Permeability and particle rejection size were found to range between 1.21 - 1.89 ×10−14 m2, and 5.8 - 4.8 μm, while tortuosity was found to decrease with pyridine concentration from 6.0 - 3.9. Consequently, cell ingress into the membrane layer is unlikely. Additionally, the blended PCL/PDO membranes were shown to be non-cytotoxic irrespective of pyridine concentration as determined with NIH 3T3s in accordance with ISO 10993. Furthermore, the EHFMs produced were found to match more of the mechanical properties of a medium sized blood vessel than any of the tissue engineered vascular grafts considered in the meta-analysis in presented this thesis.
Ensuring quality by design, mathematical modelling provides rationale when making experimental design decisions and thereby avoids the adoption of trial-and-error design evolution. Although all cell growth develops as a function of environmental conditions, ECs display a metabolic phenotype unlike most other cell types. Through a meta-analysis of data in the literature, a data-driven model of EC growth and behaviour was presented. EC growth in the prototype electrospun HFM bioreactor was thereby modelled by considering two-dimensional single-phase and porous media fluid flow with the Navier Stokes equations and Brinkman equations. Furthermore, we considered nutrient transport with an advection-diffusion-reaction model. Finally, we modelled EC growth with the data-driven model previously presented. With this model, the influence of volumetric inlet flow rate and bioreactor outlet partial pressures on EC growth rate was evaluated. Of the conditions evaluated, the EC growth rate increased with the inlet media flow rate up to a maximum of 8.036E-6 𝑠−1 at 7.5E-11 𝑚3/𝑠, after which it began to decrease. However, an increase in the lumen to ECS outlet partial pressure was found to decrease the EC growth rate.
Finally, the fabrication of an electrospun HFM bioreactor was presented. HUVECs were cultured in the electrospun HFM lumen for 28 days under hydrostatic and hydrodynamic conditions. Irrespective of fluid flow conditions, by day 28 of the culture period, HUVECs were shown to have flattened, flow-aligned morphologies, reached confluency, and expressed VE-cadherin cell-cell interactions. In addition, 𝜇CT image analysis revealed that HUVEC density peaked within 5000 𝜇m of the fibre inlet, after which it declined to a plateau, and that HUVEC growth was restricted to the lumen wall of the EHFM, with no cell ingress into the membrane wall.
It was concluded that, in concert, our findings show that: electrospun HFMs may be continuously fabricated with morphological and mechanical properties akin to that of medium-sized blood vessels. The continuous production of these electrospun HFMs opens up the potential for scalable production of electrospun vascular grafts, which to date has otherwise been restricted to batch production of short sections which has otherwise in part inhibited their wide application. Additionally, electrospun HFMs could be used as a substrate for biomedical research. specifically, these membranes could potentially be incorporated into multi-modal mechanically dynamic bioreactors to facilitate membrane-bound advective mass transfer. This approach could minimize the risk of fluid shear stress disruption of the cellular niche and passive mechanical cues, thereby improving the physiological mimicry of platforms such as the humanoid bioreactor.

Keywords: electrospinning
vascular graft
tissue engineering
hollow fibre membrane
Advisor: Ye, H
Mouthuy, P
Waters, W