Fresh Grafts Research Articles

“Fresh vs dry grafting in interlay type-1 tympanoplasty”

BackgroundChronic otitis media is a common middle ear disease that leads to varying degrees of hearing loss and otorrhoea. Tympanoplasty is a common surgery used for the reconstruction of sound conductive pathways in mucosal-type COM.AimsThe study aims to compare the fresh and the dry grafting technique in interlay type-1 tympanoplasty amongst the patients of inactive mucosal type chronic otitis media.ObjectivesThe objectives of the study are to compare graft uptake rate, pre-op and post-op AB gap, residual hearing loss, and average graft epithelialization time between the fresh and the dry grafting technique in interlay type-1 tympanoplasty.MethodsA retrospective cohort study of 48 patients of COM inactive mucosal-type who underwent interlay type-1 tympanoplasty between July 2020 and March 2022.ResultsOut of 48 patients, in 23 patients, the fresh graft was used, while in the rest 25 patients, the dry graft was used. The overall (fresh + dry group) graft uptake rate at the end of three months was 97.9%. The graft uptake rate was 100% with the use of the fresh graft and 96% with the use of the dry graft. The pre-op average air-bone (AB) gap was 12.21 ± 4.23 dB in patients where the fresh graft was used and in patients where the dry graft was used. An overall pre-op average AB gap was 13.35 ± 3.81 dB. The average time for epithelialization was 24.43 ± 4.17 days with the use of the fresh graft and 49.75 ± 6.93 days with the use of the dry graft. It was also statistically significant with a P-value of 0.001.ConclusionsThe fresh graft in the interlay type-1 tympanoplasty shows higher graft uptake rates, reduced epithelialization time, and better hearing improvement compared to dry graft. However, different complication rates are similar with the use of both types of grafts.

Fresh tilapia fish skin graft treatment of severe skin burns in a cat

AbstractAn entire female cat presented with extensive third‐degree burns covering greater than 30% of total body surface. The wound was cleaned and debrided with daily dressing management to heal with second intention. Dressing applied included honey‐based product, L‐Mesitran Ointment, and silver alginate dressing, Askina Calgitrol Thin, until healthy granulation tissue had formed. Fresh tilapia skin grafts were harvested and prepared and secured onto the wound bed. Daily lavage and graft evaluation occurred, and subsequently the skin grafts were removed on Day 13 and Day 7, respectively. Wound healing was observed without any complications or adverse side effects. In conclusion, the use of fresh tilapia skin grafts proves to be a safe, innovative, low‐cost, and effective therapeutic and promising option in treatment of challenging skin burn wounds. Tilapia fish skin grafts resulted in a clinically favourable outcome in a cat with extensive third‐degree burn wounds.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

Treosulfan- Versus Busulfan-based Conditioning in Allogeneic Hematopoietic Cell Transplantation for Myelodysplastic Syndrome: A Single-center Retrospective Propensity Score-matched Cohort Study

Treosulfan has shown promise in allogeneic hematopoietic cell transplantation (HCT) for its myeloablative properties and low toxicity. In this single-center retrospective propensity score-matched cohort study we compared treosulfan- and busulfan-based conditioning in allogeneic HCT for patients with myelodysplastic syndrome (MDS). This study included 138 adults who underwent allogeneic HCT for MDS or chronic myelomonocytic leukemia (CMML) at Princess Margaret Hospital, Toronto 2015-2022. Using propensity score matching, we compared transplant outcomes between two well-matched cohorts who received conditioning with either fludarabine-treosulfan (FT) (n=46) or fludarabine-busulfan with total body irradiation (FBT200) (n=92). A scoring system based on patient age, Karnofsky performance score and hematopoietic cell transplant comorbidity index was used to assign patients based on fitness to low-dose (30 g/m2) or high-dose (42 g/m2) treosulfan: 32 (69.6%) received high-dose treosulfan. The racial composition of the two groups was similar, with 27.2% and 21.7% of FBT200 and FT recipients, respectively, identifying as non-Caucasian (P=0.61). Primary outcomes were analyzed at a median follow-up of 747 days. Of all participants, 116 (84.0%) received graft-vs-host disease (GVHD) prophylaxis with post-transplant cyclophosphamide (PTCY) and anti-thymocyte globulin (ATG) . Patients who received FT had a superior 2-y overall survival (OS) compared to those who received FBT200: 66.9% (95% confidence interval (CI): 46.1-81.2) vs. 44.5% (95% CI: 34-54.4), hazards ratio (HR): 0.43, 95% CI: 0.22-0.84 (P=0.013). In multivariate analysis (MVA), only the use of fresh grafts (P=0.02) and FT (P=0.01) were associated with improved OS. FT was associated with superior 2-y relapse-free survival (RFS) compared to FBT200: 63.1% (95% CI: 42.6-77.9) vs. 39.1% (95% CI: 29.1-49.1), HR: 0.44 (95% CI: 0.24-0.81), P=0.008. In MVA, the use of fresh grafts (P=0.03) and FT (P=0.009) were associated with improved RFS. Recipients of FT demonstrated superior 2-y graft versus host disease relapse-free survival (GRFS) compared to those who received FBT200: 57.4% (95% CI: 37.8-72.8) vs. 35.1% (95% CI: 25.5-45). In MVA, only FT was associated with superior GRFS (P=0.02). FT recipients exhibited markedly superior 1-y event-free survival (EFS) compared to recipients of FBT200 in univariate analysis (40.3% (95% CI: 25.9-54.2) vs. 9.2% (95% CI: 4.4-16.3), HR: 0.47 (95%CI: 0.30-0.72), P<0.001) and MVA (P=0.004). FT was associated with lower 1-y non-relapse mortality (NRM) compared to FBT200 in univariate analysis (9.9% (95% CI: 3.0-21.8) vs. 29.7% (95% CI: 20.6-39.3), HR: 0.41 (95% CI: 0.17-0.96), P=0.04) and MVA (P=0.04). Our study utilized propensity score matching to demonstrate superiority of treosulfan- over busulfan-based conditioning in stem cell transplantation of patients with MDS and is the first to evaluate the performance of treosulfan-based conditioning in combination with ATG and PTCY. As such, it contributes to the increasing body of evidence supporting the safety of treosulfan, even at the dose of 42 g/m2.

Pros and Cons of Cryopreserving Allogeneic Stem Cell Products.

The COVID-19 pandemic has precipitously changed the practice of transplanting fresh allografts. The safety measures adopted during the pandemic prompted the near-universal graft cryopreservation. However, the influence of cryopreserving allogeneic grafts on long-term transplant outcomes has emerged only in the most recent literature. In this review, the basic principles of cell cryopreservation are revised and the effects of cryopreservation on the different graft components are carefully reexamined. Finally, a literature revision on studies comparing transplant outcomes in patients receiving cryopreserved and fresh grafts is illustrated.

Fresh Versus Frozen Meniscal Allograft Transplant: Revisit or Redundant? A Systematic Review

Background: Fresh-frozen allografts are the current standard in meniscal allograft transplant (MAT) surgery, due to their availability, ease of preservation, and affordability. However, fresh-frozen grafts are associated with several clinical challenges such as graft shrinkage and extrusion, among many others. Purpose: To present the current knowledge on the use of fresh meniscal allografts, presenting whether benefits associated with fresh grafts provide sufficient evidence to support their use in clinical practice. Study Design: Systematic review; Level of evidence, 5. Methods: A comprehensive search was conducted with keywords listed below. After an initial screening on title and abstract, full-text articles were assessed with the inclusion criteria. Results: A total of 78 studies matched the inclusion criteria. Literature and preclinical studies indicated that fresh meniscal allografts are beneficial for maintaining mechanical properties, graft ultrastructure, and matrix metabolism due to the presence of viable cells. Therefore, fresh allografts may address common complications associated with fresh-frozen MAT. To overcome challenges associated with both fresh-frozen and fresh allografts, a group has studied treating fresh-frozen allografts with a cell-based injection therapy. Conclusion: Fresh meniscal allografts pose several challenges including limited availability, demanding preservation procedures, and high costs. Although the role of viable cells within meniscal allografts remains controversial, these cells may be vital for maintaining tissue properties.

New strategies for sterilization and preservation of fresh fish skin grafts

The introduction of fish skin as a biological dressing for treating burns and wounds holds great promise, offering an alternative to existing management strategies. However, the risk of disease transmission is a significant concern. Therefore, this study aimed to examine how established sterilization and preservation procedures affected fish skin grafts' microbiological and histological properties for long-term usage. Lyophilization of the fish skin graft followed by rehydration in normal saline for 15 min did not change the collagen content. Furthermore, gamma irradiation of the lyophilized fish skin graft at different lengths 5, 10, and 25 KGy showed a significant reduction in microbial growth (aerobic bacteria, aerobic yeasts, and fungi) at 15- and 30 days after the irradiation. However, exposure to 10 KGy was found to be the most effective intensity among the different gamma irradiation lengths since it preserved the collagen fiber content and intensity in the lyophilized fish skin grafts at 15- and 30 days after the irradiation. These findings provide efficient preservation and sterilization methods for long-term usage of the fresh Tilapia skin grafts used for biological dressings.

The Impact of Cryopreservation on Hematopoietic Stem Cell Engraftment and Post-transplant Outcome During the COVID-19 Pandemic.

The COVID-19 pandemic has had a significant impact on the current management of allotransplanted patients in whom fresh hematopoietic stem cells (HSCs) were replaced by cryopreserved ones. The aim of the study was to determine the efficacy and safety of cryopreserved HSCs when compared with the fresh ones. A retrospective analysis of 254 allogeneic stem cell transplantations (HSCT) procedures performed between 2020-2021 included the following donors: matched related (MRD; n=68), matched unrelated (MUD; n=148) and haploidentical (HID; n=38). 50% of patients (non-cryo group) received fresh grafts, whereas the remaining patients (cryo group) were transplanted with cryopreserved cells. No differences in terms of median days to neutrophil [MRD/MUD/HID cryo- and non-cryo groups: 17 vs. 16 (p=0.27), 19 vs. 18 (p=0.83), 22 vs. 22 (p=0.83) days, respectively] and platelet [MRD/MUD/HID cryo- and non-cryo groups: 14 vs. 14 (p=0.25), 17 vs. 17 (p=0.33), 21 vs. 19 (p=0.36) days, respectively] engraftments were demonstrated. Among MUD graft recipients, platelet engraftment rates were 81% in the cryo- and 96% in the non-cryo group (p=0.01). OS rates were comparable at 1 year after HSCT between MRD/MUD/HID cryo- and non-cryo groups: 53% vs. 60% (p=0.54), 60% vs. 66% (p=0.5), 50% vs. 41% (p=0.56), respectively. During the COVID-19 pandemic, cryopreserved HSCs did not have a negative impact on median engraftment time and OS when compared to fresh HSCs. In the MUD group, platelet engraftment rate was lower in cryopreserved HSC recipients.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p&amp;lt;0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p&amp;lt;0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p&amp;lt;0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p&amp;lt;0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p&amp;lt;0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Complications and Failures of Autologous Heterotopic Cranial Bone versus Alloplastic Cranioplasties.

Fresh autologous cranial bone graft has traditionally been regarded as the ideal cranioplasty material; however, long-term comparisons of outcomes with modern alloplastic materials are absent in the literature. The authors evaluated complications and failures among cranioplasties performed with fresh, heterotopic, cranial bone graft versus 3 common alloplastic materials. Random-effects meta-analyses of logit-transformed proportions were performed on studies published between 1971 and 2021 to evaluate complications and failures of cranioplasties performed with fresh, autologous, heterotopic cranial bone; polyetheretherketone (PEEK); polymethylmethacrylate (PMMA); or titanium with a mean follow-up of 12 months or more. Generalized mixed model meta-regressions were performed to account for heterogeneity and to evaluate the contributions of moderators to outcomes variables. A total of 1490 patients (mean age, 33.9 ± 10.8 years) were included. Pooled, all-cause complications were 6.2% for fresh, heterotopic, autologous cranial bone (95% CI, 2.1% to 17.0%; I2 = 55.0; P = 0.02), 18.5% for PEEK (95% CI, 14.0% to 24.0%; I2 = 0.0%; P = 0.58), 26.1% for titanium (95% CI, 18.7% to 35.1%; I2 = 60.6%; P < 0.01), and 28.4% for PMMA (95% CI, 12.9% to 51.5%; I2 = 88.5%; P < 0.01). Pooled all-cause failures were 2.2% for fresh autologous cranial bone (95% CI, 0.4% to 10.6%; I2 = 0.0%; P = 0.45), 6.3% for PEEK (95% CI, 3.2% to 12.3%; I2 = 15.5%; P = 0.31), 11.4% for titanium (95% CI, 6.7% to 18.8%; I2 = 60.8%; P < 0.01), and 12.7% for PMMA (95% CI, 6.9% to 22.0%; I2 = 64.8%; P < 0.01). Meta-regression models indicated that each alloplastic subtype significantly and independently predicted higher complications, whereas titanium and PMMA were significant predictors for all-cause failures compared with autologous bone. All 3 subtypes were predictive of higher cranioplasty failures secondary to infection compared with autologous bone. Cranioplasties performed with fresh, autologous, heterotopic cranial bone grafts resulted in lower complication and failure rates compared with alloplastic materials.

Cryopreservation of Growth Factor-Mobilized Peripheral Blood Stem Cells Does Not Compromise Major Outcomes after Allogeneic Hematopoietic Cell Transplantation: A Single-Center Experience

During the COVID-19 pandemic, cryopreservation of allogeneic donor stem cell products ensured availability of products at the start of conditioning for hematopoietic cell transplantation (HCT). Following recommendations from unrelated donor registries, including the National Marrow Donor Program (NMDP), many centers started to cryopreserve related donor peripheral blood stem cell (PBSC) products. Throughout the process, several centers have published outcomes with cryopreserved versus fresh products, some with conflicting results. Even though cryopreservation was initially considered only a temporary measure driven by the pandemic, potential advantages include greater flexibility of transplant timing. However, concern about detrimental effects of cryopreservation including increased risks of graft rejection, relapse and consequently mortality remained. The primary objective was to describe our center's experience comparing outcomes following PBSC transplantation with cryopreserved versus fresh grafts. This was an observational case study with retrospective review comparing cryopreserved grafts (N=213), to a recent historical cohort (controls) using fresh grafts (N=167). In multivariable analyses, the adjusted hazard ratio (fresh vs. cryo) we for overall mortality was HR= 1.20 (95% confidence interval (CI), 0.79-1.82; p=0.40), for non-relapse mortality HR=0.99 (95% CI, 0.55-1.77; p=0.98), and for relapse HR=0.94 (95% CI, 0.60 -1.48; p=0.80). The adjusted hazard ratio for platelet engraftment was HR=1.31 (95% CI, 1.05-1.63; p=.02) and the odds ratio of grades III-IV acute GVHD was OR=1.75 (95% CI 1.01-3.04; p=.05) with fresh compared to cryopreserved grafts. There was no demonstrable difference in the risk of chronic GHVD. Although longer-term follow-up is needed, these data provide preliminary reassurance that in the event of another pandemic or should the logistical need arise in individual patients, cryopreservation of PBSC products constitutes a reasonably safe alternative.

Choosing Between Older Matched Sibling Donor and Younger Matched Unrelated Donor in Allogeneic Hematopoietic Cell Transplantation: Comparison of Clinical Outcomes in Acute Myeloid Leukemia and Myelodysplastic Syndrome

The choice between an older matched sibling donor (MSD) and a younger matched unrelated donor (MUD) in allogeneic hematopoietic cell transplantation (HCT) remains a subject of ongoing debate. In this single-center retrospective study of 377 patients who received peripheral blood stem cell (PBSC) transplants for acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), we compared outcomes of 85 patients who received grafts from MSDs age >60 years and 292 patients who received grafts from MUDs age <30 years. Compared to recipients of MSD transplants, recipients of MUD transplants were younger and more likely to receive dual T cell depletion (TCD), a higher CD34+ cell dose, and a fresh graft. Recipients of MSD transplants were maintained on immunosuppressive therapy longer than those who received MUD grafts. We found no differences in overall survival, relapse-free survival, graft-versus-host disease (GVHD)-free and relapse-free survival, nonrelapse mortality, relapse, engraftment, graft failure, and acute GVHD between recipients of MSD grafts and recipients of MUD grafts. We report a higher 30-day incidence, but not 1-year incidence, of bloodstream infections among recipients of MUD transplants compared to subjects who received their grafts from a MSD. The incidence of moderate-severe chronic GVHD was higher in MSD graft recipients compared with MUD graft recipients in univariate analysis, but not in multivariate analysis. Although this difference could reflect the greater use of dual TCD, known to be associated with very low rates of chronic GVHD in MUD transplant recipients, the incidence of moderate-severe chronic GVHD was no different between MSD and MUD transplant recipients following propensity score matching, suggesting that other variables could be responsible. Taken together, our data suggest that in patients with AML or MDS who receive PBSC transplants, such factors as convenience, ease of access, and costs should be considered when selecting an older MSD over a younger MUD.

Treatment of Chondral Lesions in the Knee.

Articular cartilage injuries are common and lead to early joint deterioration and osteoarthritis. Articular cartilage repair techniques aim at forming a cartilaginous neo-tissue to support the articular load and prevent progressive degeneration. Several techniques are available for this purpose, such as microfracture and chondrocyte transplantation. However, the procedural outcome is often fibrocartilage, which does not have the same mechanical resistance as cartilaginous tissue. Procedures with autologous osteochondral graft have a morbidity risk, and tissue availability limits their use. As such, larger lesions undergo osteochondral transplantation using fresh or frozen grafts. New techniques using minced or particulate cartilage fragments or mesenchymal stem cells are promising. This paper aims to update the procedures for treating chondral lesions of the knee.

Clinical impact of cryopreservation of allogeneic hematopoietic cell grafts during the onset of the COVID-19 pandemic

AbstractAt the onset of the COVID-19 pandemic, the National Marrow Donor Program mandated the cryopreservation of hematopoietic cell grafts from volunteer unrelated donors because of numerous patient and donor safety concerns and logistical hurdles. Using the Center for International Blood and Marrow Transplant Research outcomes database, we report the impact of cryopreservation on overall survival (OS) and other outcomes within 1 year after hematopoietic cell transplantation (HCT). We analyzed 1543 recipients of cryopreserved allografts receiving HCT at US centers during the first 6 months of the pandemic and compared them with 2499 recipients of fresh allografts during a 6-month period in 2019. On multivariable regression analysis, we observed no difference in the OS (P = .09), nonrelapse mortality (P = .89), graft-versus-host disease (GVHD), or GVHD- and relapse-free survival (P = .58) in recipients of cryopreserved vs fresh allografts. Disease-free survival (DFS) was lower in the cryopreserved allograft recipients (P = .006) because of a higher risk of relapse (P = .01) compared with the fresh allograft recipients. Primary graft failure was higher (P = .01), and the risk of chronic GVHD was lower (P = .001) with cryopreservation compared with fresh grafts. In conclusion, although there was no negative impact of cryopreservation on OS, relapse was higher, and DFS was lower than that with no cryopreservation. Fresh grafts are recommended as the pandemic-related logistical hurdles resolve. Cryopreservation should be considered an option for patients when fresh grafts are not feasible.

Exosomes Derived from Human Adipose-Derived Stem Cells Cannot Distinctively Promote Graft Survival in Cryopreservation Fat Grafting.

Cryopreserved fat has limited clinical applications due to its rapid absorption, high degree of fibrosis, and risk of complications after grafting. Many studies have verified that Adipose-derived mesenchymal stem cell-derived exosomes (ADSC-Exos) can improve fresh fat graft survival. This study assessed whether ADSC-Exos could improve the survival of cryopreserved fat grafts. Exosomes were isolated from human ADSCs were subcutaneously engrafted with adipose tissues stored under different conditions (fresh; cryopreserved for 1 month) into the backs of BALB/c nude mice (n = 24), and exosomes or PBS were administered weekly. Grafts were harvested at 1, 2, 4, and 8 weeks, and fat retention rate, histologic, and immunohistochemical analyses were conducted. At 1, 2, and 4 weeks after the transfer, cryopreserved fat grafts in groups of exosome-treated showed better fat integrity, fewer oil cysts, and reduced fibrosis. Further investigations of macrophage infiltration and neovascularization revealed that those exosomes increased the number of M2 macrophages at 2 and 4 weeks (p<0.05), but had limited impact on vascularization (p>0.05). It's important to note that no significant differences (p>0.05) were observed between the two groups in both histological and immunohistochemical evaluations at 8 weeks post-transplantation. This study suggests that ADSC-Exos could improve the survival of cryopreserved fat grafts in the short term (within 4 weeks), but the overall improvement was poor (after 8 weeks). This suggests that the utility of using ADSC-Exos to treat cryopreserved adipose tissue grafts is limited. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Mechanical Properties of Fresh, Frozen and Vitrified Articular Cartilage.

Osteochondral allograft transplantations are typically used to treat focal articular cartilage injuries where the damaged cartilage is replaced with fresh cadaveric donor grafts. Despite the notable success rate of this procedure, it is limited by fresh donor tissue availability which can only be stored for approximately 28days after harvest. Vitrification, a form of cryopreservation, can extend the storage time of cartilage. Although it has shown to preserve chondrocyte viability, its effect on the mechanical properties of the tissue has not been thoroughly investigated. Therefore, in this study, the mechanical properties of fresh, frozen, and vitrified articular cartilage were evaluated through unconfined compression testing. Results showed that the peak modulus, equilibrium modulus, and relaxation time constants of the vitrified and control samples (tested one day after harvest) were similar and higher than the fresh (tested 21days after harvest) and frozen samples. This demonstrated that vitrification does not adversely affect the mechanical properties of cartilage and can be used as an alternative to fresh allografts which are limited by storage time. The fresh samples also had inferior mechanical properties compared to the control samples suggesting that vitrified allografts could potentially improve clinical outcomes in addition to increasing donor tissue availability.

Cryopreservation of Allogeneic Hematopoietic Cell Products During COVID-19 Pandemic: Graft Characterization and Engraftment Outcomes

The COVID-19 pandemic triggered the deployment of unfamiliar measures to safeguard successful allogeneic hematopoietic cell transplantation (allo-HCT). Among these measures, cryopreservation offered logistical benefits that could outlast the pandemic, including graft availability and timely clinical service. The purpose of this study was to evaluate graft quality and hematopoietic reconstitution in patients transplanted with cryopreserved allogeneic stem cell products during the COVID-19 pandemic. We evaluated 44 patients who underwent allo-HCT using cryopreserved grafts consisting of hematopoietic progenitor cells (HPC) apheresis (A) and bone marrow (BM) products at Mount Sinai Hospital. Comparative analyses of 37 grafts infused fresh during the one-year period preceding the pandemic were performed. Assessment of cellular therapy products included total nucleated cell and CD34+ cell enumeration, viability, and post-thaw recovery. The primary clinical endpoint was the evaluation of engraftment (absolute neutrophil count [ANC] and platelet count) and donor chimerism (presence of CD33+ and CD3+ donor cells) at day +30 and +100 post-transplant. Adverse events related to cell infusion were also analyzed. Patient characteristics were comparable between the fresh and cryopreserved groups with 2 exceptions in the HPC-A cohort: the number of patients in the cryopreserved group that received haploidentical grafts was 6 times that in the fresh group, and the number of patients in the fresh group with a Karnofsky performance score >90 was double that in the cryopreserved group. The quality of HPC-A and HPC-BM products was not affected by cryopreservation, and all grafts met the release criteria for infusion. The pandemic did not affect the time between collection and cryopreservation (median, 24 hours) and time in storage (median, 15 days). Median time to ANC recovery was significantly delayed in recipients of cryopreserved HPC-A (15 vs 11 days, P=.0121), and there was a trend toward delayed platelet engraftment (24 vs 19 days, P=.0712). The delay in ANC and platelet recovery was not observed when only matched graft recipients were compared. Cryopreservation did not affect the ability of HPC-BM grafts to engraft and reconstitute hematopoiesis, and there was no difference in the rates of ANC and platelet recovery. Achievement of donor CD3/CD33 chimerism was not affected by cryopreservation of either HPC-A or HPC-BM products. Graft failure was observed in only 1 case, a recipient of cryopreserved HPC-BM. Three recipients of cryopreserved HPC-A grafts died before ANC engraftment from infectious complications. Remarkably, 22% of our studied population had myelofibrosis, and almost half received cryopreserved HPC-A grafts with no graft failure observed. Finally, patients receiving cryopreserved grafts were at a higher risk of infusion-related adverse events than those receiving fresh grafts. Cryopreservation of allogeneic grafts results in adequate product quality with minimal impact on short-term clinical outcomes, except for an increased risk of infusion-related adverse events. Cryopreservation is a safe option in terms of graft quality and hematopoietic reconstitution with logistical benefits, but additional data are needed to determine long-term outcomes and assess whether this is a suitable strategy for at-risk patients.

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes. MethodsTransplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit–granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 6/kg, between 6 and 8 × 106/kg and <6 × 106/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes. ResultsOver 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2–positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 108, with a median viability of 76%. The median CD34+ cells/kg was 5 × 106, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 108/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 106/kg and 276.5 × 104/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 106/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 106 CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 106 CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 6/kg at collection showed a significantly lower TNC and CD34 yield. ConclusionsTransplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.

Chick Chorioallantoic Membrane as a Patient-Derived Xenograft Model for Uveal Melanoma: Imaging Modalities for Growth and Vascular Evaluation

Patient-derived tumor xenografts (PDXs) have emerged as valuable preclinical in vivo models in oncology as they largely retain the polygenomic architecture of the human tumors from which they originate. Although animal models are accompanied by cost and time constraints and a low engraftment rate, PDXs have primarily been established in immunodeficient rodent models for the in vivo assessment of tumor characteristics and of novel therapeutic cancer targets. The chick chorioallantoic membrane (CAM) assay represents an attractive alternative in vivo model that has long been used in the research of tumor biology and angiogenesis, and can overcome some of these limitations. In this study, we reviewed different technical approaches for the establishment and monitoring of a CAM-based uveal melanoma PDX model. Forty-six fresh tumor grafts were acquired after enucleation from six uveal melanoma patients and were implanted onto the CAM on ED7 with Matrigel and a ring (group 1), with Matrigel (group 2), or natively without Matrigel or a ring (group 3). Real-time imaging techniques, such as various ultrasound modalities, optical coherence tomography, infrared imaging, and imaging analyses with Image J for tumor growth and extension, as well as color doppler, optical coherence angiography, and fluorescein angiography for angiogenesis, were performed on ED18 as alternative monitoring instruments. The tumor samples were excised on ED18 for histological assessment. There were no significant differences between the three tested experimental groups regarding the length and width of the grafts during the development period. A statistically significant increase in volume (p = 0.0007) and weight (p = 0.0216) between ED7 and ED18 was only documented for tumor specimens of group 2. A significant correlation of the results for the cross-sectional area, largest basal diameter, and volume was documented between the different imaging and measurement techniques and the excised grafts. The formation of a vascular star around the tumor and of a vascular ring on the base of the tumor was observed for the majority of the viable developing grafts as a sign of successful engraftment. The establishment of a CAM-PDX uveal melanoma model could elucidate the biological growth patterns and the efficacy of new therapeutic options in vivo. The methodological novelty of this study, investigating different implanting techniques and exploiting advances in real-time imaging with multiple modalities, allows precise, quantitative assessment in the field of tumor experimentation, underlying the feasibility of CAM as an in vivo PDX model.

316 - Cryopreservation for Allogeneic Hematopoetic Stem Cell Transplants Is Non-Inferior to Fresh Graft Products

316 - Cryopreservation for Allogeneic Hematopoetic Stem Cell Transplants Is Non-Inferior to Fresh Graft Products