Bioengineered tissue and cell therapy products are efficiently cryopreserved with pathogen-inactivated human platelet lysate-based solutions

Abstract

Background There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products. Methods We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary. Results Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at − 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at − 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs. Conclusions Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation

“…Previous reports have compared canine mesenchymal stem cells stored in cryopreservation solution for approximately 1 year with those stored unfrozen and showed comparable survival rates, cell markers, and functionality [11]. In humans, the viability and functionality of platelets frozen in sheet form for 18 months do not differ from those of non-frozen platelets [12]. These results are consistent with our results, indicating that the cells retain viability, functionality, and differentiation capacity after cryopreservation.…”

“…In this study, we confirmed that porcine MNs can be preserved for 2 years using proper storage procedures. Cell cryopreservation studies have shown that canine and human adipose tissues, platelets, pancreatic cells [8][9][10][11][12], and even small organs, such as rat kidneys, can be cryopreserved [13]. Although intracellular ice crystal formation presents a challenge in the cryopreservation process, it can be averted by adequate immersion in a suitable freezing solution and replacement of water content.…”

Comparison between Platelet Lysate, Platelet Lysate Serum, and Fetal Bovine Serum as Supplements for Cell Culture, Expansion, and Cryopreservation

“…There are different protocols and studies on cryopreservation based both on xeno-free commercial solutions and on human platelet lysate in different cell types, mainly in MSCs from the umbilical cord, bone marrow, and adipose tissue. These have shown that DMSO combined with PL in different concentrations maintains cell viability and proliferation after post-thawing recovery and that PL can function as a substitute for FBS in cryopreservation solutions [42][43][44][45], a conclusion similar to that which can be inferred from the results of this study.…”