Cryopreservation of human lung tissue for 3D ex vivo analysis

Abstract

Ex vivo culture techniques have assisted researchers in narrowing the translational gap between the lab and the clinic by allowing the study of biology in human tissues. In pulmonary biology, however, the availability of such tissues is a limiting factor in experimental design and constrains the reproducibility and replicability of these models as scientifically rigorous complements to in vitro or in vivo methods. Cryopreservation of human lung tissue is a strategy to address these limitations by generating cryopreserved biobanks of donors in the ex vivo study of pulmonary biology. Modern cryopreservation solutions, incorporating blends of cryoprotective extracellular macromolecules and cell-permeant non-toxic small molecules, have enabled the long-term storage of human lung tissue, allowing repeated experiments in the same donors and the simultaneous study of the same hypothesis across multiple donors, therefore granting the qualities of reproducibility and replicability to ex vivo systems. Specific considerations are required to properly maintain fundamental aspects of tissue structure, properties, and function throughout the cryopreservation process. The examples of existing cryopreservation systems successfully employed to amass cryobanks, and ex vivo culture techniques compatible with cryopreservation, are discussed herein, with the goal of indicating the potential of cryopreservation in ex vivo human lung tissue culture and highlighting opportunities for cryopreservation to expand the utility of ex vivo human lung culture systems in the pursuit of clinically relevant discoveries.

Keywords: 3D culture; Cryopreservation; Ex vivo; Human lung culture; Tissue culture.

Fig. 1

Diagram illustrating the cryopreservation of human precision-cut lung slices for cryobank generation and experimental use. The thin nature of precision-cut lung slices enables the mass storage of several slices from the same donor in a multitude of cryoprotectants. Figure illustrations were generated in BioRender

Fig. 2

Diagram illustrating the sample preparation process for ex vivo lung microexplants. With the appropriate cryoprotectants, tissue microexplants can be up to approximately 0.5 cm³ in volume. Figure illustrations were generated in BioRender

Fig. 3

Diagram illustrating possible experimental protocols used for experiments involving decellularized lung. Tissues cryopreserved prior to decellularization can still be used in experiments as whole tissues or subjected to alternate decellularization protocols, while cryopreserved decellularized scaffolds are less adaptable for other protocols but a more rigorous model as all thawed scaffolds from a single donor would have undergone the same decellularization process. Figure illustrations were generated in BioRender.

Fig. 4

Diagram illustrating the cryopreservation of lung organoids in various stages of growth or preparation. Lung tissue fragments can be used to directly derive stem cells or instead cryopreserved and used later as a source of stem cells. These stem cells can either be cryopreserved or used to develop organoids, which can then either be used in experiments or cryopreserved themselves for convenient use at a later date. Figure illustrations were generated in BioRender