Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy

Abstract

Background aimsMesenchymal stromal cells (MSCs) are being extensively researched for cell therapy and tissue engineering. We have engineered MSCs to express the pro-apoptotic protein tumor necrosis factor–related apoptosis inducing ligand (TRAIL) and are currently preparing this genetically modified cell therapy for a phase 1/2a clinical trial in patients with metastatic lung cancer. To do this, we need to prepare a cryopreserved allogeneic MSCTRAIL cell bank for further expansion before patient delivery. The effects of cryopreservation on a genetically modified cell therapy product have not been clearly determined.MethodsWe tested different concentrations of dimethyl sulfoxide (DMSO) added to the human serum albumin ZENALB 4.5 and measured post-thaw cell viability, proliferation ability and differentiation characteristics. In addition, we examined the homing ability, TRAIL expression and cancer cell–killing capacities of cryopreserved genetically modified MSCs compared with fresh, continually cultured cells.ResultsWe demonstrated that the post-thaw viability of MSCs in 5% DMSO (v/v) with 95% ZENALB 4.5 (v/v) is 85.7 ± 0.4%, which is comparable to that in conventional freezing media. We show that cryopreservation does not affect the long-term expression of TRAIL and that cryopreserved TRAIL-expressing MSCs exhibit similar levels of homing and, importantly, retain their potency in triggering cancer cell death.ConclusionsThis study shows that cryopreservation is unlikely to affect the therapeutic properties of MSCTRAIL and supports the generation of a cryopreserved master cell bank.

89Zr-oxine labelling and PET imaging shows lung delivery of a cell/gene cancer therapy

“…To assess 89 Zr-oxine cytotoxicity, freshly harvested MSCTRAIL were radio-labelled over a 10-fold range of doses between 1515 and 152 kBq/10 6 cells (as measured after 3 washes), or sham labelled in PBS alone, or PBS + 3% DMSO (used as a 89 Zr-oxine vehicle). A 20 minute labelling incubation was chosen to facilitate use within the 90 minutes post-defrosting during which TRAIL-MSCs typically maintain maximum viability when left in the cryopreservant [20]. Labelling efficiency correlated negatively with 89 Zr-oxine dose (R 2 =0.804), with the lowest two doses achieving the highest labelling efficiency with between 29 and 33% retained after 3 washes (Figure S2A), comparable to prior reports using a 30 minute incubation [12].…”

“…Cryopreserved MSCTRAIL doses are thawed immediately prior to patient transfusion in the TACTICAL trial [20], and will be radio-labelled between thawing and transfusion for the imaging cohort. For 89 Zr doses equivalent to 37 to 100 MBq per patient receiving a cell dose of 5×10 6 cells/kg (4×10 8 cells for a patient of 80 Kg), we assessed the effects of labelling MSCTRAIL doses immediately after defrosting encompassing the clinical range of 92.5-250 kBq/10 6 cells.…”

“…PET signal correlated with viable cell signal from bioluminescence imaging, increasing confidence in the reliability of this technique. Labelling was achieved from frozen cell stocks with relevant doses of 89 Zr-oxine within 45 minutes - below the 90 minutes over which frozen MSCTRAIL retain optimal viability post-thawing [20], suiting this approach to translation.…”

Genetically modified mesenchymal stromal cells in cancer therapy

“…Although most of the clinical trials using allogeneic MSCs have thawed the therapeutic product at the bedside, there has been some discussion in the literature that cryopreservation can affect certain key therapeutic characteristics of the cell product. We have already modified our cryopreservation procedure to ensure that our product is not affected, but this should be checked for any cell and gene therapy product that requires freezing [109].…”

Selaginella bryopteris Aqueous Extract Improves Stability and Function of Cryopreserved Human Mesenchymal Stem Cells

“…Deployment of intracellular agents has also notable cytotoxic side effects, for instance, lethality associated with the cooling and thawing processes [ 10 ]. Over the time, the striking challenges faced with the use of currently available cryoprotectants have led to the search of alternate measures [ 11 – 14 ].…”