Cytotherapy

Background and AimGamma delta T cells are multivalent immune cells with innate and adaptive features that sense a broad spectrum of tumor-associated stress patterns to lyse them without prior potentiation. They are therefore a good option for developing an off-the-shelf therapeutic immune cell product with antitumor functions. Therefore, we aimed to develop an optimized cGMP compatible protocol for expanding V gamma 9-V delta 2 T cells in a serum-free media for adoptive immunotherapy applications. Methods and ResultsPeripheral blood mononuclear cells (PBMC) from healthy donors were activated with Zoledronic Acid (ZOL) and IL-2 for 14 days. Cell proliferation, fold expansion, and phenotype were monitored. Among the regular donors, the baseline levels (day 0) of CD3+Vgamma9+delta2+ T cells were (05.10+/-0.74%). There was a robust expansion of V gamma 9-V delta 2 T cells in the serum-free media (110+/-29.89-fold). During processing, an abrupt reduction of {beta} T cells was observed as early as day +07. After two weeks, 87.82+/-5.11% (n=8) of T cells were CD3+Vgamma9+ in Optimizer with 97.15+/-0.7% of the CD3+Vgamma9+ T cells positive for delta2. The CD3+Vgamma9+ NKG2D+ increased during expansion, reaching 93.76+/-1.55% expression on day +14. V gamma 9-V delta 2 T cells expanded in the serum-free (Optimizer) media had relatively reduced variance and reduced over all yield and fold expansion but comparable percentage of Vgamma9+ and {beta} TCR+ T cells on day 14. A flow cytometry-based tumor-toxicity assay gauged the antitumor functions against K562 cell lines. Pretreatment of K562 cells with ZOL differentially enhanced (2.48+/-0.76 fold) the cytotoxic capacity of V gamma 9-V delta 2 T cells in a donor-dependent manner. The conditions for lentiviral transduction and transgene expressions of V gamma 9-V delta 2 T cells were improvised as gauged by GFP expression driven by CMV promoter (39.09+/- 8.94%) without compromising the viability. ConclusionWe have optimized a cGMP compatible protocol for expansion of human V gamma 9-V delta 2 T cells in a serum-free media with high purity and viability in as early as 07 days. Pretreatment of target tumor cells with ZOL enhanced the cytotoxicity, revealing the impact of V gamma 9-V delta 2 T cell intrinsic factors in tumor lysis. This protocol, may be scaled up for clinical translation for adoptive immunotherapy.

Apheresis, a conventional starting point for manufacturing chimeric antigen receptor (CAR) T-cell therapy, poses challenges due to the length and invasiveness of the procedure, the high demand for and limited quantity of apheresis beds, and additional resource constraints at collection centers. Furthermore, traditional CAR T-cell manufacturing often involves extended cell culture periods, leading to a final product that has progressed through the differentiation process and contains a higher frequency of cells with phenotypes that are indicative of lower functionality or potency.1 Here, we show that anti-CD19 CAR T-cells manufactured from fresh whole blood with minimal ex vivo expansion using Ingenui-T exhibit comparable or superior CAR-mediated and CD19-dependent functional activity compared to CAR T-cells manufactured from cryopreserved leukapheresis material following the a conventional manufacturing process. Anti-CD19 CAR T-cell production, manufactured from whole blood in less than 3 days of in vitro culture using Ingenui-T, yielded an average of about 40 million cells per 100 mL with high CD3 purity and viability. Furthermore, the final product was composed of cells with less differentiated phenotypes and sustained cytotoxic activity against CD19+ target cells at a lower dose than conventionally manufactured CAR T cells in preclinical in vitro assays. Ingenui-T is an innovative vein-to-vein solution, aimed at enhancing the patient experience, feasibility, and accessibility of CAR T-cell therapy by alleviating challenges linked to apheresis-based methods, with its patient-friendly nature, cost-effectiveness, and distinctive methodology.

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify priming conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e. priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-{gamma}) and Tumor Necrosis Factor alpha (TNF-) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-{gamma}/TNF- priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors e.g. PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry-based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER(R) EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC.

Mesenchymal stromal cells (MSCs) are currently being tested in numerous clinical trials as potential cell therapies for the treatment of various diseases and due to their potential immunomodulatory, pro-angiogenic, and regenerative properties. However, variabilities in tissue sources, donors, and manufacturing processes and the lack of defined critical quality attributes (CQAs) and clinically relevant mechanism of action (MoA) pose significant challenges to identify MSC cell therapy products with a predictable therapeutic outcome. This also hinders regulatory considerations and broad clinical translation of MSCs. MSC products are often administered to the patient immediately after thawing from cryopreserved vials (out-of-thaw). However, the qualifying quality-control assays are either performed before cryopreservation, or after culturing the post-thaw cells for 24-48 hours (culture-rescued), none of which represent the out-of-thaw product administered to patients. In this study, we performed a broad functional characterization of out-of-thaw and culture-rescue MSCs from bone marrow (BM-MSCs) and cord tissue (CT-MSCs) using macrophage activation and T cell proliferation-based in vitro potency assays and deep phenotypic characterization using single-cell RNA-sequencing. Using this data, we developed unbiased computational models, specifically symbolic regression (SR) and canonical correlation analysis (CCA) models to predict the immunomodulatory potency of MSCs. Overall, our results suggest that manufacturing conditions (OOT vs. CR) have a strong effect on MSC-function on MSC interactions with macrophages and T cells. Furthermore, single-cell RNA-seq analyses of out-of-thaw BM and CT-MSCs indicate a tissue of origin-dependent variability and heterogeneity in the transcriptome profile. Using symbolic regression modeling we identified specific single-cell transcriptomic attributes of MSCs that predict their immunomodulatory potency. In addition, CCA modeling predicted MSC donors with high or low immunomodulatory potency from their transcriptome profiles. Taken together, our results provide a broad framework for identifying predictive CQAs of MSCs that could ultimately help in better understanding of their MOAs and improved reproducibility and manufacturing control of MSCs.

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

Acute graft versus host disease (GVHD) remains a significant complication following hematopoietic stem cell transplant (HSCT), despite improved human leukocyte antigen (HLA) matching and advances in prophylactic treatment regimens. Previous studies have shown promising results for future regulatory dendritic cell (DCreg) therapies in the amelioration of GVHD. This study evaluates the effects of cryopreservation on DCreg generation, generation of young and older DCreg in serum-free media, and the feasibility of DCreg generated from young and older HSCT donor monocytes. DCreg were generated in X-vivo 15 serum-free media from donor monocytes. Donors included young and older individuals, either healthy donors or HSCT patients. Phenotypic differences in cell populations were assessed via flow cytometry while pro-inflammatory and anti-inflammatory cytokine production was evaluated in culture supernatants. The number of DCreg generated from cryopreserved monocytes of healthy donors was not significantly different from freshly isolated monocytes. DCreg generated from cryopreserved monocytes had similar levels of co-stimulatory molecule expression, inhibitory molecule expression, and cytokine production as freshly isolated monocytes. Young and older healthy donor monocytes generated similar numbers of DCreg with similar cytokine production and phenotype. Although monocytes from older HSCT patients produced significantly fewer DCreg, DCreg from young and older HSCT patients have a comparable phenotype and cytokine production. Monocytes from young and older myelodysplastic syndrome (MDS) patients generated reduced numbers of DCreg compared to non-MDS monocytes. Results suggest cryopreservation of monocytes from many HSCT patients allows for cost effective generation of DCreg for the prevention and treatment of GVHD on an as needed basis. Although generation of DCreg from MDS patients require further assessment, these data support the possibility of in vitro generated DCreg as a therapy to reduce GVHD-associated morbidity and morbidity in young and older HSCT recipients.

BackgroundAdipose-derived mesenchymal stem cells (ADSCs) have emerged as a promising therapeutic modality for cellular therapy because of their rapid proliferation and potent cellular activity compared to conventional bone marrow-derived mesenchymal stem cells (MSCs). Cosmetic lipoaspirates provide an easily obtainable source of ADSCs. Cryopreservation facilitates their clinical application due to increased transportability and pooling of sufficient numbers of cells. However, proper cryopreservation techniques have not been established yet.\n\nMethodsWe evaluated the post-thaw viability and ADSC functions after cryopreservation with three cryoprotectants (serum containing 10% dimethylsulfoxide (DMSO), serum-free: CP-1TM, DMSO-free: SCB-DFTM) at two temperature (-80{degrees}C, -150{degrees}C) and two cell densities: (1 x 106, 7 x 106 cells/mL) for up to 18 months using cryovials. After determining optimal conditions, we also tested if large quantities of ADSCs remained viable after 18 months of cryopreservation in a 100-mL cryobag. Rate-controlled freezing methods or liquid nitrogen storage were not exploited.\n\nResultsADSCs cryopreserved in serum containing 10% DMSO or CP-1TM at -150{degrees}C and 7 x 106 cells/mL were most viable (>85%) after 18 months without perturbation of MSC functions. Even suboptimal conditions (-80{degrees}C, 1 x 106 cells/mL, no DMSO) assured >80% viability when stored for up to 9 months. Large quantities of ADSCs in a cryobag were properly cryopreserved.\n\nConclusionsA programmable freezer or liquid nitrogen storage is not necessary. CP-1TM is preferable in terms of side effects. Simplified cryopreservation methods (-80{degrees}C and no DMSO) can be used for up to 9 months, resulting in reduced infusion toxicities and lower costs.

Differentiation of induced pluripotent stem cells (iPSC) into monocytes, monocyte-derived macrophages (MDM), and monocyte-derived dendritic cells (moDC) represents a powerful tool for studying human innate immunology and developing novel iPSC-derived immune therapies. Challenges include inefficiencies in iPSC-derived cell cultures, labor-intensive culture conditions, low purity of desired cell types, and feeder cell requirements. Here, a highly efficient method for differentiating monocytes, MDMs, and moDCs that overcomes these challenges is described. The process utilizes commercially-available materials to derive CD34+ progenitor cells that are apically released from a hemogenic endothelium. Subsequently, the hemogenic endothelium gives rise to highly pure (>95%), CD34-CD14+ monocytes in 19-23 days and yields 13.5-fold more monocytes by day 35 when compared to previous methods. These iPSC-monocytes are analogous to human blood-derived monocytes and readily differentiate into MDM and moDC. The efficient workflow and increase in monocyte output heightens feasibility for high throughput studies and enables clinical-scale iPSC-derived manufacturing processes.

During the COVID-19 pandemic, there was a disruption in the supply of the widely used erythroid differentiation media product, Iscoves Modified Dulbeccos Medium, manufactured by Biochrom AG. IMDM is a critical component of ex vivo erythropoiesis protocols used to generate genetically modified red blood cells for the study of malaria host factors. Therefore, we set out to identify the best alternative IMDM product for efficient erythroid differentiation of hematopoietic stem cells into enucleated red blood cells for use in our research. We tested other IMDM products, including I2911 (from Millipore Sigma), P04-20450 (from Pan Biotech) and EP-CM-L0216 (from ElabScience Technologies) which are all marketed specifically as replacements for the Biochrom AG product. We found that while FG0465, I2911, and P04-20450 were all sufficient for ex-vivo erythropoiesis, FG0465 IMDM was superior to other products tested in supporting maximal erythroid cell proliferation and enucleation.

Mesenchymal Stromal Cells (MSC) face several heterogeneity challenges hindering clinical and commercial success. Employing a multiple response model, interplay between donor heterogeneity, and critical processing parameters (CPPs), effects on MSC potency and cell expansion attributes were investigated through computed composite attribute scores. Twelve unique CPP combinations were tested in thirteen marrow-derived MSC(M) and five adipose-tissue MSC(AT) training and test datasets, respectively. Donor heterogeneity and select CPP conditions affected a curated gene panel (surrogate for MSC potency); while MSC expansion was primarily influenced by CPPs. Model performances were evaluated against clinical effectiveness data from a previously deployed clinical trial; top-performing model predicted donor rankings coincided with clinical effectiveness data, validating the modeling approach used. Our model predicted that only 8% of tested donors were agnostic to CPPs; a majority (62%) of donors showed CPP-dependent optimal composite quality attributes, with MSC seeding density as a key driver; medium supplementation and oxygen preferences were highly donor dependent. Approximately 30% of donors performed poorly at all conditions tested and may be prospectively identified using a subset of genes (TGFB, VEGF, PDCD1LG1, PDCD1LG2, IDO). Model predicted optimal parameters worked for 69% of tested donors, while sub-optimal parameters worked for only 23% of donors and were confirmed in an independent CD14+ macrophage assay. Our integrated computational and experimental framework predictably identified interactive effects of donor heterogeneity and CPP conditions to optimize MSC potency attributes.

In vitro expansion of spermatogonial stem cells (SSCs) has been established using animal-derived fetal bovine serum (FBS) and bovine serum albumin (BSA). However, the use of animal components during cell culture introduces the risk of contaminating cells with pathogens, and leads to animal epitope expression, rendering them unsuitable for medical use. Therefore, this study set out to develop a xeno-free, fully defined media for the expansion of human SSCs. We show that the molecules Prostaglandin D2 (PGD-2) and Insulin-Like Growth Factor 1 (IGF-1) can replace FBS and BSA in cell culture media without loss of viability or expansion capability, and that Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitor Y-27632 supplementation improves viability after cryopreservation. Long-term SSC cultures expanded in xeno-free, defined culture conditions shared identical protein expression profiles for well-known SSC markers, while gene expression analyses revealed a significant improvement in quiescent SSC and pan-germ markers. This xeno-free, defined formulation allows for standardized SSC culture free of animal pathogens.

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80{degrees}C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 {micro}m. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

Myeloid progenitor cell (MPC) therapy can protect against infection and can be used as an irradiation countermeasure, but can also prevent rejection of matched grafts. Here we report on the matching requirements between MPCs and skin grafts, and on the development of a non-lethal preconditioning model that supports MPC-induced protection of skin grafts. Mouse MPCs (mMPC) were obtained from HSC following 10-day ex vivo expansion. Skin grafts were tested in host mice that either received a lethal dose of irradiation and reconstitution with allogeneic mMPC and third party HSCs or were given injections with depleting antibodies and chemotherapeutics followed by transplantation of mMPC without HSC. mMPC-matched grafts were protected and no significant difference was observed between mice receiving irradiation and fully matched grafts (major and minor transplantation antigens), partially matched grafts (major only), and fully matched grafts in mice that received sublethal irradiation. Haploidentical grafts (half of the MHC alleles not matched at all) are not protected. We conclude that closely matched mMPCs are sufficient to prevent rejection. We also show that mMPCs are effective in a trachea transplant model. Graft protection does not require either lethal preconditioning or an accompanying HSC transplantation, and affects both T- and B-cell responses.

This article describes a stem cell line derived by reprogramming of native human islet cells that consistently generates pure populations of endocrine pancreatic clusters following a simple differentiation protocol. Surprisingly, the population of stem cell derived pancreatic endocrine clusters that was most consistently capable of regulating blood glucose in rodent models of diabetes lacked robust expression of the key beta cell maturation-associated factor NKX6-1 but did manifest high expression of other key drivers of endocrine cell specification and maturation, ISL1 and MAFA. These data support the hypothesis that multiple pancreatic profiles can be identified in stem cell derived cultures and that these have disparate in vivo potency. The population with low NKX6-1 and high in vivo potency was further characterized by transcriptome profiling as an endocrine-committed population progressively maturing in vitro to a state proximal to the native islet.

With advancements in genetic diagnostics and genotype-based therapeutics, the demand for germline genetic testing in post-hematopoietic stem cell transplantation patients is increasing. Due to genetic chimerism, blood samples can no longer be used for germline testing after transplantation. This study aims to identify the most suitable tissue for germline analysis following stem cell transplantation by investigating alternative tissue sources. Buccal swab, eyebrow hair, and nail samples were analyzed for donor-derived DNA using next-generation sequencing and short tandem repeat analysis, with linear regression used for evaluation. Factors such as HLA match, transplantation type, sex, and time after transplantation were also evaluated for their effect on donor-derived DNA share. Buccal swab and nail samples exhibited 25% and 22% higher proportions of donor-derived DNA compared to eyebrow hair follicles, respectively. The median donor DNA share in eyebrow hair follicles was 1% for NGS and 3% for STR. Factors such as matched related donors, higher HLA match, different donor-recipient sex, and longer time post-transplantation correlated with lower donor DNA shares. Eyebrow hair follicles are a promising tissue for accurate germline genetic testing in post-SCT patients. Patient characteristics like donor relatedness, HLA match, sex match, and time after transplantation should be considered.

{gamma}{delta} T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are {gamma}{delta} T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than {gamma}{delta} T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of V{delta}2+ T cells, while V{delta}1+ T cells likewise have anti-tumor potential. Here, we investigated whether {gamma}{delta} T cells directly isolated from blood could be efficiently expanded while maintaining function. {gamma}{delta} T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure {gamma}{delta} T cells. Isolated V{delta}1+ and V{delta}2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. After expansion, potential effector functions of {gamma}{delta} T cells were demonstrated by IFN-{gamma}, TNF- and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure {gamma}{delta} T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, {gamma}{delta} T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

Patients in need of a hematopoietic stem cell transplant frequently have pre-existing donor-specific antibodies (DSA) which can impair engraftment. These patients often require intensified conditioning regimens, even in the setting of partially matched (semi-allogeneic) donors. Reducing the toxicity of conditioning and desensitization protocols is therefore a major goal. We previously showed that enzymatic desensitization of donor-specific IgG using imlifidase and EndoS improved murine bone marrow engraftment in donor sensitized, autoimmune-prone recipient mice. Conditioning included a 6 Gy total body irradiation, cyclophosphamide, bortezomib, and T cell depletion. In non-autoimmune prone semi-allogeneic recipients sensitized to the donor, we demonstrate that desensitisation with imlifidase and EndoS permits long-term donor bone marrow engraftment and induces tolerance to subsequent allogeneic skin grafts under a 3 Gy irradiation protocol. Enzymatic inactivation of DSA facilitates donor hematopoietic stem cell engraftment in allo-sensitized semi-allogeneic recipients in a reduced intensity conditioning protocol.

BackgroundVascularized composite allotransplantation has become a viable reconstructive option for selected patients, but preservation remains a major barrier to broader clinical application. Static cold storage is the current gold standard, yet ischemia reperfusion injury and limited preservation times restrict its effectiveness. Recent advances in machine perfusion and subzero non-freezing storage (or supercooling) have prompted renewed interest in optimizing graft viability. MethodsFollowing PRISMA guidelines, we systematically searched PubMed, EMBASE, and Cochrane, covering studies published from June 2022 through August 2025 for studies on ex vivo preservation of vascularized composite allotransplantations. Eligible articles included original studies in English evaluating postharvest, pretransplant preservation strategies. Data extracted were study design, preservation methods, perfusates, and primary outcomes. Risk of bias was assessed using SYRCLE for animal studies and JBI for human/cadaver studies. ResultsSeventeen studies met inclusion criteria: one on static cold storage, thirteen on machine perfusion, and three on supercooling. Static cold storage research has declined, with the only recent study investigating sub-normothermic machine perfusion as a recovery adjunct. Machine perfusion studies focused on optimization of perfusion parameters, perfusate composition, and circuit design. Red blood cell-based perfusates remained common, but alternative oxygen carriers such as polymerized hemoglobin-based oxygen carrier-201 and dextran oxygen microcarriers showed promise despite edema-related challenges. Supercooling studies demonstrated feasibility of multi-day preservation in rodent and porcine models. Overall, risk of bias was high or unclear across animal studies, mainly due to selection and performance bias, whereas the single human ex vivo study showed low risk of bias. ConclusionsThe field of vascularized composite allograft preservation is expanding rapidly, with machine perfusion and supercooling emerging as the most promising strategies to extend graft viability beyond the limits of static cold storage. However, translation to clinical setting remains limited by small preclinical studies, methodological heterogeneity, and the paucity of functional and immunologic endpoints. Standardized protocols, robust large-animal models, and eventual human feasibility trials are needed to establish clinically applicable preservation strategies. Level of evidence: IV

Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDX) in immunodeficient NSG mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.