Effectively Improve the Astaxanthin Production by Combined Additives Regulating Different Metabolic Nodes in Phaffia rhodozyma

Astaxanthin is an important natural resource that is widely found in marine environments. Metabolic regulation is an effective method for improving astaxanthin production in Phaffia rhodozyma. Most studies have focused on single regulators, which have limited effects. In this study, 16 metabolic regulators were screened to improve astaxanthin production in high-yield and wild-type strains. Fluconazol and glutamic acid increased astaxanthin volumetric yield in MVP14 by 25.8 and 30.9%, respectively, while ethanol increased astaxanthin volumetric yield in DSM626, 29.3%. Furthermore, six additives that inhibit the competing pathways and promote the main pathway for astaxanthin synthesis were selected for combination treatment. We found that the optimal combination was penicillin, ethanol, triclosan, and fluconazol, which increased astaxanthin cell yield by 51%. Therefore, we suggest that simultaneously promoting the master pathways (mevalonate) and inhibiting competing pathways (fatty acid synthesis and ergosterol) is the best strategy to improve astaxanthin cell yield. Moreover, regulators of the biomass pathway should be avoided to improve cell yield. This study provides a technical basis for the utilisation of astaxanthin in P. rhodozyma.

The Donor – Recipient Weight Ratio is a Reliable Marker for Cell Yield in Hematopoietic Stem Cell Donations

Bone marrow transplants remain an import source of hematopoietic stem cells for patients suffering from specific diseases like aplastic anemia, for pediatric patients with malignant and non-malignant blood cell disorders, and for situations in which graft-versus-host disease (GvHD) is a concern. Identifying the optimal donor to achieve a 3-5 x 108/kg of recipient weight TNC yield may be challenging. In an analysis of 687 consecutive donors, donor and procedure characteristics were related to TNC/kg of recipient weight using Spearman correlation coefficients as well as linear and multiple regression analysis. We found correlations between donor WBC (r = 0.17), donor platelet counts (r = 0.15), donor BMI (r = 0.10), and the percentage of donor estimated blood volume accessed for harvesting (r = -0.57) with TNC/kg of recipient weight. The strongest correlation existed between the donor-recipient weight ratio and the TNC/kg (r = 84). In a multivariate regression analysis, the donor-recipient weight ratio influenced the TNC/kg of recipient weight more significantly (adjusted R2 = 0.84) than all other related variables put together. The minimal donor-recipient weight ratio associated with a TNC/kg of at least 3x108/kg of recipient weight was 0.8 (mean 3.425; 95% CI 2.01, 5.8). Using this donor-recipient ratio provides national bone marrow donor registries with a practical and simple measure to assure optimal cell yield outcomes in hematopoietic stem cell donations.

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering

Objective The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Design Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity Results Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. Conclusions The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

The Impact of Pre-Apheresis Health Related Quality of Life on Peripheral Blood Progenitor Cell Yield and Donor's Health and Outcome: Secondary Analysis of Rdsafe and BMT CTN 0201

Introduction: The adverse events associated with hematopoietic stem cell donation have been extensively studied. There is an increasing literature linking psychological factors including stress, anxiety and depression to higher levels of inflammatory burden leading to poorer post-procedural outcomes including longer hospital stays and increased pain perception. Here, we aimed to evaluate whether pre-donation health related quality of life (HRQoL) markers predict toxicity profile and stem cell yield following stem cell donation in healthy donors. Methods: The study population included adult granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell (PBSC) related donors (RD) (n= 157) and unrelated donors (URD) (n=179) who were enrolled in Related Donor Safety Study (RDSafe) and Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0201 clinical trials. Pre-donation HR QoL was assessed using the Short-Form (SF-8) in RDSafe and SF-12 questionnaire in BMT CTN 0201 (higher score is better). Pain and toxicity were collected on study specific forms. The primary outcome was the incidence of skeletal pain on day 5 of G-CSF administration. The secondary outcomes were the incidence of skeletal pain and highest toxicity level across selected body symptoms at 1 month, 6 months and 1-year post-donation. Another secondary outcome included CD34+ per liter of blood processed (x10 6/L) on day 5 of G-CSF as a measure of collection yield. The association between pre-apheresis HRQOL measures and pain and acute toxicities was characterized using means and SDs and compared using the t-test. Association between HRQoL and cell yield was assessed using the Pearson correlation coefficient. RD and URD were analyzed separately. Results: URDs were younger than RDs (median age 35 vs. 63). A higher proportion of RDs were female (50% vs. 40%) and obese (41% vs. 35%). A higher proportion of RD PBSC donations required 2 days or more (44% vs 21%). More RDs were collected with lower volume procedures (<18L, 28% vs. 16%), and required a central line (28% vs. 11%). RDs were more likely to report pre-donation grade 1-2 pain (27% vs. 8%) and other toxicities (16% vs. 6%). The mean pre-donation physical component summary (PCS) and mental component summary (MCS) score of RDs were 54.5 (SD 7.0) and 55.1 (SD 5.8), respectively . In the univariate analysis (table 1), pre-donation lower PCS score of RDs was associated with significantly more grade 2-4 pain at 1 month (p=0.0038) and 1-year post-donation (p=0.0099) (Table 1). In multivariable analysis (table 2), pre-donation PCS remained significantly associated with grade 2-4 pain 1-month post-donation (p=0.0098). More specifically, RDs with pre-donation PCS scores in the higher quartile were less likely to experience pain compared with donors with PCS scores in the lower quartile (OR 0.1; 95% CI 0.01-0.83; p=0.005). There was also a trend toward increased grade 2-4 pain at 1-year post-donation among RDs with lower PCS score (p=0.0176). Other outcomes such as pain at day 5 of G-CSF, other toxicities at day 5 of G-CSF, 1 month, 6 months and 1-year post-donation were not associated with pre-donation PCS score. Similarly, there was no significant association between RD pre-donation MCS score and collection-related symptoms at any time point. The mean pre-donation PCS and MCS scores of URDs were 56.2 (SD 4.7) and 54.5 (SD 5.5), respectively . In a univariate analysis, there was no association between PCS score or MCS score and donation associated pain and toxicities at any time point post-donation. Due to low event rates, multivariable analysis was not performed in the URD setting. Based on the multivariable regression analysis, there was no correlation between pre-apheresis HRQoL score (PCS or MSC) and PBSC collection yield in either the RD or URD setting. Conclusion: Our study demonstrates that pre-donation QoL markers are significantly associated with the toxicity profile after PBSC donation in the RD setting as adult RD with lower pre-donation physical QOL experience increased levels of pain after a PBSC collection procedure. There were no such associations found in URD in this small sample. Our findings may help clinicians to identify donors at higher risk of pain with donation, and lead to personalized information and interventions (e.g. increased analgesia) for specific donors. Future study with a larger sample is required to validate the results. Figure 1 Figure 1. Disclosures Farhadfar: Incyte: Consultancy. Stefanski: Novartis: Honoraria. Pulsipher: Equillium: Membership on an entity's Board of Directors or advisory committees; Adaptive: Research Funding; Jasper Therapeutics: Honoraria. Shaw: mallinkrodt: Other: payments; Orca bio: Consultancy.

Recruitment of NK Cells during Autologous Apheresis Allows Efficient NK Cell Collection for Immunotherapy

Background: Natural killer (NK) cell-based immunotherapies are emerging as a new, cutting-edge, promising cancer treatment. NK cells have been used to generate autologous as well as allogenic chimeric antigen receptor NK (CAR-NK) cells, and are currently being tested in multiple clinical trials. The advantage of these cells over CAR-T cells is that they show a superior toxicity profile, less on-target/off-tumor effects, and their toxicity mechanism could be independent of CAR. Additionally, allogenic CAR-NK cells are less immunogenic and cause less graft versus host disease in comparison to CAR-T cells. NK-cell collection during apheresis is the first step in the production of CAR-NK-cells. Efficient collection of NK-cells is paramount to successful manufacture of a CAR-NK cell product. Here, we present our single-center experience on individualized high-flow autologous lymphocyte apheresis in heavily pre-treated patients who qualified for a CAR-T treatment. In this abstract, we demonstrate data on NK-cell collections that derived from our research on T-cell collections. Study design and methods: In this retrospective, descriptive study, we compared collection efficiencies (CE%), absolute efficiencies (AE; absolute yield) and relative efficiencies (RE; AE/total number of absolute circulating pre-apheresis cells) for NK- and T-cell collections. Recruitment of cells was assumed if RE>1. The analysis was performed for 9 heavily pre-treated diffuse large B-cell lymphoma patients who underwent autologous T-cell collections for CAR-T treatment between October 2018 and November 2019. These 9 patients were chosen because the data on pre-apheresis NK- and T-cell concentrations, and harvest NK- and T-cell yield, were available for them. For calculation of CE% a standard formula was used (Figure 1, A). Results: A total of 9 high-flow lymphocyte collections achieved the cell yield of 1x10 9 cells for T- cells and 88% (n=8) achieved the cell yield of 1x10 9 cells for NK-cells. The median pre-apheresis cell concentration for NK-cells was 0.21 x10 9/L (range 0.05 - 0.29) and for T-cells was 0.69 x10 9/L (0.11 - 2.09). The median average blood flow was 77 ml/min (49 - 132), the median processed blood volume was 13.51L (7.35 - 31.17), while the median ratio of processed blood volume to total blood volume was 2.57 (1.46 - 6.67). The median AE for NK-cells with 1.58x10 9 (0.55 - 5.9) was markedly lower but not statistically significant (p=0.14) than that of T-cells with 5.5x10 9 (1.88 - 18.41). The CE of NK-collections was median 89% (29 - 125), while the CE of T-cell collections was median 68% (31 - 93) and they were not statistically different (p=0.605). The same was observed for RE which was 3.04 (0.63 - 4.31) for NK-cells and 1.91 (0.68 - 4.52) for T-cells with no statistically significant difference (Independent-Samples Mann-Whitney U Test, p=0.836). Figure 1 presents the differences in AE (B), CE (C) and RE (D) between NK- and T-cell collections. Conclusions: During high-flow lymphocyte apheresis, NK- as well as T-cells are recruited, as in the majority of collections the cell yield is greater than the absolute circulating pre-apheresis cell count (when RE was > 1). Thus, the origin and immunophenotype of these NK- and T-cells should be subject of further investigation. CE for NK- and T-cells are similar and high enough to perform efficient cell collections of both cell types for immunotherapy. Figure 1 Figure 1. Disclosures Jalowiec: Kite Pharma Gilead Sciences': Honoraria, Speakers Bureau. Daskalakis: Amgen: Other: Travel grant; Roche: Other: Travel grant; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Celgene/BMS: Consultancy, Other: Travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; NovoNordisk: Other: Travel grant.

Significant Plasma Cell Loss (up to 90%) Despite Preserved Overall Cell Viability - Time Dependent Changes Observed in Multiparametric Flow Cytometry Analysis of Bone Marrow Samples in Multiple Myeloma

Multiparametric flow cytometry (MPFC) is a mainstream laboratory method used in the diagnosis of multiple myeloma. Minimal residual disease (MRD) assessment by EuroFlow next-generation flow cytometry allows assessment down to an assay sensitivity of 1x10 -5. Delayed sample processing remains a common challenge due to logistical limitations. Specialized tests performed in central pathology laboratories are frequently located a considerable distance from healthcare providers. Our study aims to evaluate the impact of delayed sample processing on plasma cell yield and bone marrow sample stability. There is little published data available. Plasma cell yield and bone marrow sample stability were investigated in patients with multiple myeloma who underwent bone marrow biopsy. Participants were included based on ³10% plasma cell burden by morphological quantification on the bone marrow aspirate smear. Bone marrow aspirates were collected in EDTA (with three samples also collected in lithium heparin) and stored at four degrees Celsius. Samples were analyzed by MPFC within four hours of collection, at 24 and at 48 hours after collection. CD138 and CD38 co-expression were used to identify plasma cells, and absence of 7-AAD to determine cell viability. Mean fluorescence intensity (MFI) of CD138 and CD38 was recorded. Statistical analyses were performed using two-tailed Wilcoxon signed-rank tests and repeated measures ANOVA with significance assigned at p<0.05. Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p<0.001) while sample integrity remained unchanged (p>0.05). The most marked reduction in plasma cell detection was seen between initial processing and 24 hours (median absolute reduction 9%, range 0 to 23% and median relative reduction 37%, range -8 to 90%, p<0.01). Further significant reduction of plasma cells occurred after an additional 24 hours (p=0.025). At 48 hours, the median absolute reduction in plasma cell yield from initial testing was 12% (range 1 to 24%) and median relative reduction was 40% (range 18 to 90%). Sample integrity remained constant. The median viability at collection, 24 hours and 48 hours was 91%, 93% and 95% respectively. The most significant specimen deterioration observed was 13% viability reduction to 75% overall by 48 hours. Three of the participants had additional samples collected in lithium heparin anticoagulant media that were analyzed in parallel with their EDTA samples. Plasma cell yield remained similar across the two different anticoagulants with overall cell viability remaining high in lithium heparin (³90%). A trend of time-dependent reduction of CD138 MFI was observed with lithium heparin but not with EDTA. This study demonstrates the significance of time to processing as a pre-analytical variable in MPFC in multiple myeloma. The greatest loss of plasma cells occurs within the first 24 hours after collection but continues to fall significantly out to 48 hours. Reductions of up to 90% were observed in our small cohort and represent a potential 1 log reduction in yield. This decrease in plasma cell yield raises questions of reliability and validity of flow cytometry, whereby the sensitivity depth may be compromised if the sample cannot be processed on the same day of collection. It is a technical limitation of flow cytometry in comparison to polymerase chain reaction methods where sensitivity is unaffected by delays in processing. The overall viability of cells within the samples remained stable over time, despite the decline in plasma cells. A reduction in CD138 MFI is observed in lithium heparin storage medium that may impact on standardized gating techniques. Further validation studies are warranted to explore these phenomena. MRD monitoring in multiple myeloma is rapidly becoming an accepted standard of care in the evaluation of treatment response and represents an independent prognostic maker of progression free survival that can be used to guide further therapy. Our findings indicate the potential of false negative MRD results with delays in sample processing. This questions the current consensus guidelines that recommend samples can be processed up to 2 days after collection. These guidelines may need to be revised in the near future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A Robust and Highly Efficient Approach for Isolation of Mesenchymal Stem Cells from Wharton’s Jelly for Tissue Repair

BackgroundMesenchymal stem cells (MSCs) derived from umbilical cord Wharton’s Jelly (WJ-MSCs) are emerging as promising therapeutics for a variety of diseases due to their ability of regeneration and immunomodulation and their non-tumorigenic and non-immunogenic properties. Although multiple protocols have been developed for WJ-MSCs isolation, insufficient cell numbers, heterogeneous cell population, and variations in procedures between different laboratories impede further clinical applications.MethodsWe compared six widely used WJ-MSCs isolation methods regarding cell morphology, yield, purity, proliferation rate, and differentiation potential. Based on these analyses, we developed a new isolation approach called “Mince-Soak-Digest (MSD)”, and compared its efficiency with the existing methods. Furthermore, we transplanted WJ-MSCs isolated by different methods to the rat uterus to test their ability for tissue repair.ResultsBased on the comparison and analysis of the six widely used isolation protocols, we identified that the inefficiency of the digestion of the extracellular matrix results in low cell yield. Thus, we have developed a robust and highly efficient method to isolate MSCs from WJ by incorporating a soaking step to facilitate the digestion of the extracellular matrix and release of the cells. Our newly developed method generates significantly higher cell yield (4 to 10-fold higher) than six widely used methods that we tested with high purity and consistency. Importantly, by transplantation of WJ-MSCs isolated by MSD to the rat uterus, we repair the endometrial injury and restore the fertility of the rats.ConclusionOur results provide a robust and highly efficient approach for isolating WJ-MSCs to restore injured tissue. The higher efficiency of MSD assures the abundance of WJ-MSCs for clinical applications. Furthermore, the reliability of MSD contributes to the standardization of WJ-MSCs isolation, which eliminates the discrepancies due to isolation procedures, thus facilitates the evaluation of the efficacy of WJ-MSCs across various human clinical applications.