Rubinstein et al. Whether the unit used for the transplants was RBC-replete or RCR was not considered among the variables associated with the engraftment- and transplantation-related events. For the sake of convenience, we will call these first generation 1st Gen cord blood processing techniques. It should be noted that these methods are often erroneously referred to as red cell depletion when all of these techniques retain considerable RBCs and do not deplete, but only reduce the number of RBCs.
This is of concern because it is well-known that the success of CBT is critically dependent on cell dose, and insufficient cell dose is widely regarded as the most important limitation for umbilical CB transplantation, especially for adults and large children [ 1 , 3 ]. Table 1 lists some of the most common 1st Gen CB processing techniques. Technical comparison of some of the most popular 1st Gen CB processing techniques and the proprietary 2nd and 3rd MaxCell CB processing technologies. To maximize CB cell dose, R.
Chow has developed two proprietary alternative CB volume reduction technologies 2nd Gen and 3rd Gen which he calls MaxCell CB processing technologies, because both share the common characteristics of 1 depleting or reducing plasma, 2 maximizing stem cell, progenitor, nucleated and mononuclear cell recovery after processing and most importantly, and 3 having identical cellular composition upon infusion for one version of the third generation technology Tables 1 — 3.
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A comparison of the volumes and amount of some of the various components of first hetastarch , second and third generation CB technologies. Plasma is removed or reduced from the product prior to the cryopreservation [ 18 ]. Although the resulting volume of CB products processed by such a method is two to three times larger, 2nd Gen results in greater than True nucleated cell loss was less than 0. Such minimal loss was reproducible from validation to an actual MaxCell inventory of more than 12, CB products that had pre- and post- processing CBC available.
Even after thawing, in post-thaw segment samples, a median of While the processing methods and cryopreserved products are distinctly different, with the preferred embodiment of 3rd Gen technology, the cellular compositions of the final infused products are virtually identical between the two MaxCell technologies. Comparisons between HES 1st Gen and MaxCell 2nd and 3rd Gen are performed using parallel processing comparison using one half of the same cord blood unit for first generation hetastarch and the other half for MaxCell CB Products [ 18 ].
Cell types which exhibit significant differences are highlighted by boldfaced fonts. Second and third generation cord blood products have similar yield for all cell lineages, and only differ in the final RBC yield depending on the embodiment of 3rd Gen used. P values are calculated using the paired-sample t -test. Third generation CB processing technology is perhaps the most flexible CB processing technology developed to date, with the additional option to reduce RBCs without losing as much stem, progenitor, nucleated, and mononuclear cells as 1st Gen RCR techniques Table 3.
Table 1 highlights some of the processing technical differences among various popular 1st Gen techniques versus 2nd Gen and 3rd Gen CB processing technologies. While the processing methods and cryopreserved products are distinctly different, the cellular composition of the final infused product is identical between the two MaxCell technologies using the preferred embodiment of 3rd Gen Table 3 technology, while other embodiments can be adapted to reduce RBC, free hemoglobin, DMSO, and WBC lysate load for certain situations to better suit the needs of the individual patients Table 2.
Barker et al. Unfortunately, no hard data accompanied this implication or their recommendation and the authors ignored the data on tens of thousands of CB units presented by Chow et al. More importantly, the superior outcome data for the MaxCell CB products [ 8 ] were similarly disregarded. This outcome assumes that large cord blood banks have normal and similar distribution of patients, disease indications, and disease stage.
To study this issue, Chow et al. Page et al. In fact, in clinical studies of MaxCell CB products, outstanding clinical outcome in terms of engraftment and patient survival have been achieved. Ballen et al. While acknowledging some of the significant design flaws in their study, the authors concluded that automated processing systems resulted in higher day neutrophil recovery than manually processed plasma-reduced products; however, overall survival by day was not different among the three groups.
Many study decisions and design issues were not clearly explained or delineated in this study, such as patient selection or study end point selection. For patients receiving Max Cell 2nd Gen CB products and manually processed RCR CB products, there were only and patients, respectively, when hundreds to thousands more were available fitting the study criteria during the study period for both of these groups.
For example, even in a study with just pediatric hematological malignancy patients from one transplant center U. In fact, it is obvious that of the 16 public CB banks in the study, at least four of the banks each alone had more patients fitting the exact criteria for patients transplanted with manually processed CB in the study, so the reasons why these other patients were not selected and the reasons for inclusion of the particular patients in the study were not clear.
The decision to use only day survival, and not 1-year, 3-year, or 5-year overall survival, was also quite unconventional. In fact, our experience of a large MaxCell CB bank with thousands of patients transplanted with its products have shown that survival or transplant-related mortality during the first days is often not predictive for long-term survival or patient mortality [ 8 , 35 — 43 ]. The most rigorous method to address clinical outcome differences between different types of CB processing methods would be to conduct prospective double-blind placebo-controlled clinical trials; however, in HSCT, this is largely not feasible.
Instead, matched-pair analyses allow for comparisons that are reasonably free from extraneous factors. To investigate rigorously whether clinical outcome differences exist between RCR and MaxCell CB products, Chow and collaborators performed matched-pair analysis using retrospective outcome data. For both studies, all thalassemia and pediatric hematological malignancy patients were included and outcome were similar before and after matched-pair comparisons. A logistic regression model was used to find patients with similar characteristics to form pairs.
For both studies, cumulative incidence was used for ANC neutrophil and platelet 20K and 50K engraftment. Kaplan—Meier was used for overall survival, disease-free survival, transplanted-related mortality, and relapse or autologous recovery. Cox regression analysis, log-rank test, univariate comparisons and the paired Prentice-Wilcoxon method were performed to compare the two matched-pair groups.
Using the above methodologies, two rigorous retrospective matched-pair analysis of patients using RCR versus MaxCell CB products were conducted for thalassemia as well as for pediatric hematological malignancy patients [ 35 — 38 ]. Absolute Neutrophil Count ANC is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.
Outcome comparisons of the two patient groups pre-match showed superiority in overall survival, disease-free survival, and transplant-related mortality for patients transplanted with MaxCell CB. As the patients are mostly pediatric, there were no differences in median TNC between the two patient groups before MaxCell 9. Table 5 showed significant improvement in 1- to 3-year overall survival and disease-free survival and 1-year and 3-year transplant-related mortality with the use of MaxCell CB for thalassemia patients [ 36 , 38 ]. Interestingly, neutrophil engraftment, and short-term day survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products.
For the pediatric hematological malignancy matched-pair study, factors matched between the two groups were age, weight, HLA matches, TNC dose, disease, and disease status [ 35 ]. Arizona , Graham et al. Table 5 shows that for the pairs of pediatric hematological malignancy patients, 1- and 3-year overall survival, day, 1-year and 3-year transplant-related mortality, and platelet 20K and 50K engraftment were significantly improved with the use of MaxCell CB, while disease-free survival trended towards improvement [ 35 , 37 , 38 ].
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Superior outcome of the MaxCell CB patient group in pre-match comparisons were confirmed by the results seen in the matched-pair analysis. Again, neutrophil engraftment and short-term day survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products despite significant advantages in platelet engraftment, overall survival and transplant-related mortality.
Thus, MaxCell CB processing provides more efficient utilization of this valuable resource. This would seem to be particularly significant for those patients who participate in directed CB donation and private banking, because of the uniqueness of the cellular content and the importance of cell dose in outcome of HSCT. Screnci et al. Post-thaw cell loss vs.
Collection and Processing of Stem Cells
There are generally three main methods of manipulations after thaw of CB products, with many variations among different banks and transplant centers. Table 7 summarizes whether each method reduces the total amount or just dilutes the concentration of DMSO, free hemoglobin or WBC lysates from thawing, safe thaw-to-dilution and thaw-to-infusion times, as well as summarizes the advantages and disadvantages of each method.
Automated procedures using the Biosafe Sepax system can be used on all CB product types and was shown to be as effective as manual washing in terms of cell recovery and viability [ 46 ]. The CB product is thawed at bedside using the above thaw technique and immediately administered to the patient. Thaw to completion of infusion is typically completed within 10 min, with a maximum of 20 min, to avoid DMSO-induced toxicity and lysis of cells. Delay in infusion after thaw will lead to DMSO toxicity and cell lysis, resulting in loss of cell viability and release of potentially harmful cytokines, chemokines and cell debris that may potentially cause adverse events if released in sufficient amount.
Further clinical data from the St. There has been some concern that the presence of residual RBC in cryopreserved MaxCell CB may adversely affect the safety of HSCT; however, lysed RBC ghosts and free hemoglobin do not usually give rise to severe problems [ 49 ] unless a patient has compromised renal function or is on nephrotoxic drugs.
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The rare occurrences of acute renal failures with HSCT are frequently self-limiting or resolved with dialysis. While MaxCell manufacturers have always advocated direct infusion without post-thaw washing or dilution for most patients receiving MaxCell CB products, they also caution that for children, small patients, patients with compromised renal function pre-existing or iatrogenic , patients with known sensitivity to DMSO, RBC or WBC lysates, chemokines and cytokines, and, lastly, for transplant centers that cannot directly infuse MaxCell CB products within 10—20 min of thawing, post-thaw washing is indicated.
If post-thaw reconstitution or washing is to be performed, then it is of utmost importance to dilute the MaxCell CB product adequately serial dilutions three times to final minimal dilution within 10 min of thawing and to complete infusion of the washed product within 1—2 h, respectively Table 7. Chow et al. Comparisons of the three major CB product thaw methods and the various parameters, Pros and Cons. Pros and Cons of the three major CB product thaw methods [ 8 , 21 , 44 , 48 ]. Matched-pair analysis results comparing pairs of CBT patients receiving unwashed versus washed MaxCell CB products [ 56 — 59 ].
Paired Prentice-Wilcoxon test and log-rank tests were used to analyze patients forming pairs; 95 pairs malignancies and 34 pairs nonmalignant indications; relapse calculations are only for the 95 malignancy pairs. Paired Prentice-Wilcoxon test uses matched pairs of patients infused with post-thaw washed versus unwashed MaxCell CB products.
Log-rank tests used univariate analysis of previously matched patients. In , Rubinstein et al. The supernatant, containing DMSO, hetastarch if applicable , cell lysates, hemolysate including free hemoglobin , and any chemokines and cytokines released up to that point, are removed and cellular sediment is resuspended in one volume of fresh isotonic infusion solution equal to or greater than the original product volume.
For all products, this method achieves post-thaw stability in cases of prolonged thaw-to-infusion time and reduces the potential for infusion reactions, by significantly reducing the amount of DMSO, hetastarch if applicable , cell lysates, hemolysate including free hemoglobin , and any chemokines and cytokines. Moreover, according to Rubinstein et al. A number of recent studies have failed to confirm the latter observation as reviewed by Akel et al. MSCs from bone marrow BMSCs and adipose tissue-derived stromal cells ASCs obtained from three donors were culture expanded in three different commercially available hPL fulfilling good manufacturing practice criteria for clinical use.
Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. Feasibility and optimization of tumor-infiltrating lymphocytes generation from patients with pancreatic cancer J Immunother. We have optimized methods for generating pancreatic cancer-specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer. Tagged as: cell processing , cryopreservation , manufacturing. Previous post: Cells Weekly — February 21, RegenMed Digest - industry updates. Experimental bone marrow transplantation Mesenchymal Stem Cells — definition and assays.
True Blood - manufacturing blood from stem cells. Proposal for ISCT to revise the definition and minimal criteria of mesenchymal stromal cells. CD34 expression on human mesenchymal stromal cells. Notable market approvals in Impact of fetal bovine serum on toxicity of mesenchymal stromal cell infusions in humans.