Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application

Abstract

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.

Generation of a Functioning and Self-Renewing Diaphragmatic Muscle Construct

“…Despite the literature, several research papers reported the scaffold storage after lyophilization , our aim was to preserve dECM structure and architecture also after storage without the need for swelling procedure, to obtain in vitro a construct as similar as possible to the original muscle. Giving that dECM was preserved in a better condition when stored for a short period or using a cryoprotectant for longer time, as it happens for other biologic‐derived scaffolds , it means that this product can be treated as a fresh and viable tissue, and that standard GMP storage rules could be applied also to dECM . Nevertheless, the scaffolds used in this study were from rodents that, together with other important manufacturing issues (i.e., decontamination and sterilization), should be reconsidered before a possible clinical translation, possibly moving to the use of large animal model or human tissue samples .…”

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors

“…Decellularized oesophagus was obtained by adapting a previously reported technique optimized for simple tissues (skin, skeletal muscle); tubular structures (trachea, intestine); or more complex tissues (liver, lung, kidney) 7 , 26 – 29 . DET removed cellular components, avoiding antigenicity reaction, but preserved the major ECM molecules, maintaining elastin and sGAG content, distribution of collagen I and IV, laminin, and the overall multi-strata architecture.…”

Preservation over time of dried acellular esophageal matrix

“…1). The quality of the decellularization protocol to obtain esophageal tissue with absence of genetic material and cellular components was deeply investigated in our previous works [18,19,30].…”

“…Mechanical properties strictly depend on the quality of the ECM, which needs to be preserved during decellularization [18,19] and supercritical drying [30] processes. The EtOH/SC- It has to be emphasized that the tissue desiccation process that we described successfully acted as both long-term storage technique and high grade sterilization method.…”