Combined automated culture system for tubular structure assembly and maturation for vascular tissue engineering

Abstract

Development of the robust yet practical tissue culture systems for vascular grafting is one of the major challenges for biomedical engineering. The following questions should be solved in priority: (1) flexibility of the tissue reservoir allowing dynamic stretching, (2) seeding of cells and extraction of tissues should be easy and safe, (3) system must allow morphological observation in real time, (4) maintenance of metabolic activity of tissues should be performed automatically. In our study, we attempted to solve these problems designing in vivo-like culture chamber made of PDMS, and developed an integrated system with a perfusion bioreactor and a small digital microscope. We developed disposable cell chamber with following qualities: transparent, autoclave-sterilizable, non cell-adhesive, and having low autofluorescence. The polyacetal mold made it possible to prepare a chamber hosting the tissue of the desired shape and size. In our case, a porous tube made from PTFE was fixed inside the chamber and tubular cell culture space was prepared for loading of preformed cell spheroids. Perfusion of the media within the porous tube continuously supplied the nutrients and oxygen to the spheroids. Growth and fusion of the cell spheroids inside the chamber can be observed real time by the small digital microscope and analyzed retrospectively by time-laps movies. We loaded batches up to 900 goat fibroblast spheroids into the system; growth, development and morphological fusion of the spheroids were followed out for 10 days. Removal of the tissue without disturbing its structural integrity was possible, and histological analysis revealed reasonable fusion degree between spheroids, and 72.5 % cell survival rate as estimated by TUNEL staining. In conclusion, our system has the basic performance necessary for culturing cell spheroids for tissue-engineered vascular graft.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft

“…However, the stacking of dense sheets of cells disrupts the supply of nutrients and oxygen to the interior layer 12 . Micro tissue aggregation is reliable method for tissue formation in required size and we previously developed a method and the bioreactor for its implementation to form tubular tissue of arbitrary diameter and length 13 . However, it is challenging to control the self-assembly of the cells, and no one has yet been able to reproduce a physiological vessel with a correct three-layered structure by this approach.…”

Vascular implants – new aspects for in situ tissue engineering

“…The presented bioreactor allows cultivating three vessels in parallel. Baba et al [18] present a combined automated culture system for tubular structure assembly with enough space for dynamic stretching. The group achieved this by designing an in vivo-like culture chamber made of polydimethylsiloxane (PDMS).…”

“…The design of an intelligent bioreactor system must feature various sensory elements, a robust system setup and the possibility to reuse the bioreactor containment for several cultivation processes of TEVGs. Different kinds of TEVG bioreactor setups were presented over the past decades, with perfusion bioreactors as the predominantly‐used model [ 16 , 17 , 18 , 19 , 20 ] Features such as specifically compact [ 18 ] and cost‐efficient [ 20 ] designs promote high scalability.…”

Dynamic Flow Priming Programs Allow Tuning Up The Cell Layers Properties for Engineered Vascular Graft.

“…However, the stacking of dense sheets of cells disrupt the supply of nutrients and oxygen to the interior layer 12 . Micro tissue aggregation is reliable method for tissue formation in required size and we previously developed a method and the bioreactor for its implementation to form tubular tissue of arbitrary diameter and length 13 . However, it is challenging to control the self-assembly of the cells, and no one has not yet been able to reproduce a natural vessel with a correct three-layered structure by this approach.…”