Effects of Cryopreservation on Cell Metabolic Activity and Function of Biofabricated Structures Laden with Osteoblasts

Abstract

Biofabrication and maturation of bone constructs is a long-term task that requires a high degree of specialization. This specialization falls onto the hierarchy complexity of the bone tissue that limits the transfer of this technology to the clinic. This work studied the effects of the short-term cryopreservation on biofabricated osteoblast-containing structures, with the final aim to make them steadily available in biobanks. The biological responses studied include the osteoblast post-thawing metabolic activity and the recovery of the osteoblastic function of 3D-bioprinted osteoblastic structures and beta tricalcium phosphate (β-TCP) scaffolds infiltrated with osteoblasts encapsulated in a hydrogel. The obtained structures were cryopreserved at −80 °C for 7 days using dimethyl sulfoxide (DMSO) as cryoprotectant additive. After thawing the structures were cultured up to 14 days. The results revealed fundamental biological aspects for the successful cryopreservation of osteoblast constructs. In summary, immature osteoblasts take longer to recover than mature osteoblasts. The pre-cryopreservation culture period had an important effect on the metabolic activity and function maintain, faster recovering normal values when cryopreserved after longer-term culture (7 days). The use of β-TCP scaffolds further improved the osteoblast survival after cryopreservation, resulting in similar levels of alkaline phosphatase activity in comparison with the non-preserved structures. These results contribute to the understanding of the biology of cryopreserved osteoblast constructs, approaching biofabrication to the clinical practice.

Long-Term Cryostorage of Mesenchymal Stem Cell-Containing Hybrid Hydrogel Scaffolds Based on Fibrin and Collagen

“…When analyzing the literature data on the cryostorage of tissue engineering products based on cell-loaded hydrogels, it was noted that, as a rule, only the short-term storage effect on product morphologies/appearence for a period of up to 7 days was estimated [21][22][23]. At the same time, the When analyzing the literature data on the cryostorage of tissue engineering products based on cell-loaded hydrogels, it was noted that, as a rule, only the short-term storage effect on product morphologies/appearence for a period of up to 7 days was estimated [21][22][23]. At the same time, the possibility of cryostorage for up to 10 years has been demonstrated for cell products without a carrier [24,25].…”

“…When analyzing the literature data on the cryostorage of tissue engineering products based on cell-loaded hydrogels, it was noted that, as a rule, only the short-term storage effect on product morphologies/appearence for a period of up to 7 days was estimated [ 21 , 22 , 23 ]. At the same time, the possibility of cryostorage for up to 10 years has been demonstrated for cell products without a carrier [ 24 , 25 ].…”

The impact of cryopreservation in signature markers and immunomodulatory profile of tendon and ligament derived cells

“…The ability for cell machinery revival after cryopreservation has been investigated as a long‐term storage procedure for cells and tissues (Antebi et al, 2019; Hernandez‐Tapia et al, 2020). However, the impact of cryopreservation/thawing in the maintenance of stemness, tissue‐specific functionalities, and immunomodulatory cues of hTDCs and hLDCs has not been reported.…”

“…The effect of cryopreservation has been investigated in cell recovery after thawing and in functional responses with controversial outcomes. Although cryopreservation of periodontal ligament stem cells in cell sheet constructions did not alter their viability, proliferative capacities, and multi‐lineage differentiation (Li et al, 2017), other studies demonstrate that cryopreservation may compromise cell function, even if the loss of potency may be recovered with time (Hernandez‐Tapia et al, 2020).…”

A New Perspective for Bone Tissue Engineering: Human Mesenchymal Stromal Cells Well-Survive Cryopreservation on β-TCP Scaffold and Show Increased Ability for Osteogenic Differentiation

“…The methods used, however, were not conducive to clinical application. A recent pilot study of Hernandez-Tapia showed that osteoblasts have good survival rate after cryopreservation on β-TCP scaffold [18]. The equally recent study of Mutsenko et al [19] showed that MSCs could be frozen in 3D collagen-hydroxyapatite scaffold without significant loss of cell viability.…”